Treatment of fibrotic conditions

ABSTRACT

Treatment of fibrosis and fibrotic diseases, disorders, and conditions, and associated methods, compositions, formulations and articles.

RELATED APPLICATIONS

This application is a Continuation application of U.S. patentapplication Ser. No. 12/809,886, filed on Feb. 22, 2011 which is aNational Stage application under 35 U.S.C. §371 of InternationalApplication No. PCT/US2008/014021, filed on Dec. 22, 2008 which claimsthe benefit of priority to U.S. Provisional Application No. 61/008,795filed on Dec. 21, 2007. The disclosures of which are incorporated hereinby reference.

FIELD

The inventions relate to connexins and gap junctions, and to fibrosis,fibrotic conditions, and methods of treatment thereof.

BACKGROUND

The following includes information that may be useful in understandingthe present invention. It is not an admission that any of theinformation provided herein is prior art, or relevant, to the presentlydescribed or claimed inventions, or that any publication or documentthat is specifically or implicitly referenced is prior art.

In humans and other mammals wound injury triggers an organized complexcascade of cellular and biochemical events that will in most casesresult in a healed wound. An ideally healed wound is one that restoresnormal anatomical structure, function, and appearance at the cellular,tissue, organ, and organism levels. Wound healing, whether initiated bysurgery, disease, trauma, microbes or foreign materials, proceeds via acomplex process encompassing a number of overlapping phases, includinginflammation, epithelialization, angiogenesis and matrix deposition.Normally, these processes lead to a mature wound and a certain degree ofscar formation.

Fibroproliferative diseases, including the pulmonary fibrosis, systemicsclerosis, liver cirrhosis, cardiovascular disease, progressive kidneydisease, and macular degeneration, are a leading cause of morbidity andmortality and can affect all tissues and organ systems. Fibrotic tissueremodeling can also influence cancer metastasis and accelerate chronicgraft rejection in transplant recipients. Nevertheless, despite itsenormous impact on human health, there are currently no approvedtreatments that directly target the mechanism(s) of fibrosis.

Fibrosis is the abnormal accumulation of fibrous tissue that can occuras a part of the wound-healing process in damaged tissue. Examples offibrosis include liver fibrosis, lung fibrosis (e.g., silicosis,asbestosis, idiopathic pulmonary fibrosis), oral fibrosis,endomyocardial fibrosis, retroperitoneal fibrosis, deltoid fibrosis,kidney fibrosis (including diabetic nephropathy), andglomerulosclerosis. Liver fibrosis, for example, occurs as a part of thewound-healing response to chronic liver injury. Fibrosis can occur as acomplication of haemochromatosis, Wilson's disease, alcoholism,schistosomiasis, viral hepatitis, bile duct obstruction, exposure totoxins, and metabolic disorders. This formation of fibrotic tissue isbelieved to represent an attempt by the body to encapsulate injuredtissue. Liver fibrosis is characterized by the accumulation ofextracellular matrix that can be distinguished qualitatively from thatin normal liver. Left unchecked, hepatic fibrosis progresses tocirrhosis (defined by the presence of encapsulated nodules), liverfailure, and death. Endomyocardial fibrosis is an idiopathic disorderthat is characterized by the development of restrictive cardiomyopathy.In endomyocardial fibrosis, the underlying process produces patchyfibrosis of the endocardial surface of the heart, leading to reducedcompliance and, ultimately, restrictive physiology as the endomyocardialsurface becomes more generally involved. Endocardial fibrosisprincipally involves the inflow tracts of the right and left ventriclesand may affect the atrioventricular valves, leading to tricuspid andmitral regurgitation. Oral submucous fibrosis is a chronic, debilitatingdisease of the oral cavity characterized by inflammation and progressivefibrosis of the submucosal tissues (lamina propria and deeper connectivetissues). It results in marked rigidity and an eventual inability toopen the mouth. The buccal mucosa is the most commonly involved site,but any part of the oral cavity can be involved, even the pharynx.Retroperitoneal fibrosis is characterized by the development ofextensive fibrosis throughout the retroperitoneum, typically centeredover the anterior surface of the fourth and fifth lumbar vertebrae. Thisfibrosis leads to entrapment and obstruction of retroperitonealstructures, notably the ureters. In most cases, the etiology is unknown.However, its occasional association with autoimmune diseases and itsresponse to corticosteroids and immunosuppressive therapy suggest it maybe immunologically mediated. Deltoid fibrosis is a muscle disordermarked by intramuscular fibrous bands within the substance of thedeltoid muscle. These bands lead to secondary contractures that affectthe function of the shoulder joint. Scapular winging and secondaryscoliosis also may be related to this condition. Deltoid fibrosis hasbeen associated with fibrous contractures of the gluteal and quadricepsmuscles and is likely a similar process

Understanding of the cellular and biochemical mechanisms underlyingliver fibrosis has advanced in recent years (reviewed by Li andFriedman, J. Gastroenterol. Hepatol. 14:618-633, 1999). Stellate cellsare believed to be a major source of extracellular matrix in the liver.Stellate cells respond to a variety of cytokines present in the liver,some of which they also produce (Friedman, Seminars in Liver Disease19:129-140, 1999). As summarized by Li and Friedman, actual and proposedtherapeutic strategies for liver fibrosis include removal of theunderlying cause (e.g., toxin or infectious agent), suppression ofinflammation (using, e.g., corticosteroids, IL-1 receptor antagonists,or other agents that may suppress inflammation), down-regulation ofstellate cell activation (using, e.g., gamma interferon orantioxidants), promotion of matrix degradation, or promotion of stellatecell apoptosis. Despite recent progress, many of these strategies arestill in the experimental stage, and existing therapies are aimed atsuppressing inflammation rather than addressing the underlyingbiochemical processes. Thus, there remains a need in the art formaterials and methods for treating fibrosis, including liver fibrosis.

Gap junctions are cell membrane structures that facilitate directcell-cell communication. A gap junction channel is formed of twoconnexons (hemichannels), each composed of six connexin subunits. Eachhexameric connexon docks with a connexon in the opposing membrane toform a single gap junction. Gap junction channels are reported to befound throughout the body. Tissue such as the corneal epithelium, forexample, has six to eight cell layers, yet is reported to expressesdifferent gap junction channels in different layers with connexin 43 inthe basal layer and connexin 26 from the basal to middle wing celllayers. In general, connexins are a family of proteins, commonly namedaccording to their molecular weight or classified on a phylogeneticbasis into alpha, beta, and gamma subclasses. At least 20 human and 19murine isoforms have been identified. Different tissues and cell typesare reported to have characteristic patterns of connexin proteinexpression and tissues such as cornea have been shown to alter connexinprotein expression pattern following injury or transplantation (Qui, C.et al., (2003) Current Biology, 13:1967-1703; Brander et al., (2004), J.Invest Dermatol. 122:1310-20).

It has been reported that abnormal connexin function may be linked tocertain disease states (e.g. heart diseases) (A. C. de Carvalho, et al.,J Cardiovasc Electrophysiol 1994, 5 686). In certain connexin proteins,alterations in the turnover and trafficking properties may be induced bythe addition exogenous agents which may affect the level of gapjunctional intercellular communication (Darrow, B. J., et al. (1995).Circ Res 76: 381; Lin R, et al. (2001) J Cell Biol 154(4):815).Antisense technology has been reported for the modulation of theexpression for genes implicated in viral, fungal and metabolic diseases.See, e.g., U.S. Pat. No. 5,166,195, (oligonucleotide inhibitors of HIV),U.S. Pat. No. 5,004,810 (oligomers for hybridizing to herpes simplexvirus Vmw65 mRNA and inhibiting replication). See also U.S. Pat. No.7,098,190 to Becker et al. (formulations comprising antisensenucleotides to connexins). Peptide inhibitors (including mimeticpeptides) of gap junctions and hemichannels have been reported. See e.g.Berthoud, V. M. et al., Am J. Physiol. Lung Cell Mol. Physiol.279:L619-L622 (2000); Evans, W. H. and Boitano, S. Biochem. Soc. Trans.29: 606-612, and De Vriese A. S., et al. Kidney Int. 61:177-185 (2001).See also Becker and Green PCT/US06/04131 (“Anti-connexin compounds anduses thereof”).

Despite advances in the understanding of the principles underlyingfibrosis and the fibrotic process, there remains a significant unmetneed in suitable therapeutic options for treatment of fibrosis andfibrotic conditions. Such therapeutics compositions and treatments aredescribed and claimed herein.

BRIEF SUMMARY

The inventions described and claimed herein have many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this Brief Summary. It is not intended to beall-inclusive and the inventions described and claimed herein are notlimited to or by the features or embodiments identified in this BriefSummary, which is included for purposes of illustration only and notrestriction.

According to one aspect, the present invention is directed to methods ofdecreasing fibrosis in a tissue of a subject comprising identifying asubject in need of decreased fibrosis and administering to the subjectan anti-connexin polynucleotide, for example, an anti-connexin 43polynucleotide, thereby decreasing fibrosis in the tissue of a subject.In certain embodiments, an anti-connexin polynucleotide is ananti-connexin 26, 30, 31.1, 32, 36, 37, 40, or 45 polynucleotide. Inother embodiments, the anti-connexin polynucleotide is an anti-connexin30.3, 31, 40.1, or 46.6 polynucleotide.

According to one embodiment the subject is a mammal. In anotherembodiment the mammal is a human. In another embodiment, the subject isan animal or a bird. Birds include pets and poultry. Animals includeswine, cattle and sports animals and pets such as horses, dogs and cats.

According to one embodiment, the anti-connexin polynucleotide, e.g.,anti-connexin 43 polynucleotide, decreases a target connexin, e.g.,connexin 43, protein expression.

According to an embodiment, the anti-connexin polynucleotide, e.g.,anti-connexin 43 polynucleotide, is an anti-connexin oligonucleotide,e.g., an anti-connexin 43, oligonucleotide.

According to an embodiment, the anti-connexin polynucleotide, e.g.,anti-connexin 43 polynucleotide, is an antisense oligonucleotide, e.g.,a connexin 43 antisense oligonucleotide.

According to an alternate embodiment, the anti-connexin polynucleotide,e.g., anti-connexin 43 polynucleotide, is an siRNA oligonucleotide. Inanother embodiment, the anti-connexin polynucleotide, e.g.,anti-connexin 43 polynucleotide, is an RNAi oligonucleotide.

According to one embodiment of the present method, the tissue is skintissue, retinal tissue, brain tissue, nerve tissue, lung tissue, cardiactissue, kidney tissue or liver tissue. Other tissues where fibrosisoccurs in the body are also within the scope of the invention.

According to another embodiment of the method, the anti-connexinpolynucleotide, e.g., anti-connexin 43 polynucleotide, is administeredto prevent or retard, in whole or in part, fibrosis.

According to one embodiment of the method, the subject has a disease,disorder or condition selected from the group consisting of scleroderma,kidney fibrosis (including diabetic nephropathy), cardiac fibrosis (e.g.myocardial fibrosis), pulomanry fibrosis (e.g., glomerulosclerosispulmonary fibrosis, idiopathic pulmonary fibrosis, silicosis,asbestosis, interstitial lung disease and fibrotic lung disease, andchemotherapy/radiation induced pulmonary fibrosis), oral fibrosis,endomyocardial fibrosis, deltoid fibrosis, pancreatitis, inflammatorybowel disease, Crohn's disease, nodular fascilitis, eosinophilicfasciitis, general fibrosis syndrome characterized by replacement ofnormal muscle tissue by fibrous tissue in varying degrees,retroperitoneal fibrosis, liver fibrosis, liver cirrhosis, chronic renalfailure; myelofibrosis (bone marrow fibrosis), drug induced ergotism,glioblastoma in Li-Fraumeni syndrome, sporadic glioblastoma, myleoidleukemia, acute myelogenous leukemia, myelodysplastic syndrome,myeloproferative syndrome, gynecological cancer, Kaposi's sarcoma,Hansen's disease, collagenous colitis and acute fibrosis. According tothis embodiment, the scleroderma may be morphea, generalized morphea, orlinear scleroderma. Also according to this embodiment, the kidneyfibrosis may be glomerular sclerosis, renal tubulointerstitial fibrosisor progressive renal disease. Further to this embodiment, the pulmonaryfibrosis may be diffuse interstitial pulmonary fibrosis.

According to another embodiment of the method, the fibrosis is acutefibrosis. The acute fibrosis may be in response to various forms oftrauma including accidental injuries, infections, radiation orchemotherapy treatments.

According to another embodiment of the method, the fibrosis is chronicfibrosis.

The invention also includes methods for treating and/or preventing, inwhole or in part, various diseases, disorders and conditions, including,for example, capsular contracture, Dupytren's contracture, Volkmann'scontracture, Ledderhose's contracture, Peyronie's contracture orrecurrence thereof, comprising administering a effective amount of acomposition comprising an anti-connexin polynucleotide. In onembodiment, the composition is administered to the site of the injurybefore, at the time of and/or after a release procedure (e.g., forcedmanipulation, open release, arthroscopic release, or debulking of scar)to prevent the recurrence of scarred and abnormal tissue and/or furthercontracture.

According to a different embodiment, the connexin 43 is human connexin43.

In an embodiment, the anti-connexin polynucleotide, e.g., anti-connexin43 polynucleotide, inhibits intercellular communication by decreasinggap junction and/or hemichannel formation.

According to a further aspect, the patent invention is directed to amethod of decreasing or preventing fibrosis in a subject in need thereofor at risk thereof, said method comprising administering atherapeutically effective amount, e.g., an anti-fibrotic amount, of ananti-connexin polynucleotide, e.g., anti-connexin 43 polynucleotide, tosaid subject. According to one embodiment of this aspect, the methodcomprises administering a therapeutically effective amount of ananti-connexin oligonucleotide to said subject. According to an alternateembodiment, the method comprises administering a therapeuticallyeffective amount of an anti-connexin 43 oligonucleotide to said subject.In one embodiment, the anti-connexin polynucleotide is effective todownregulate connexin expression, preferably connexin 43 expression. Inanother embodiment, the anti-connexin polynucleotide or oligonucleotideis an antisense polynucleotide or oligonucleotide, e.g., an antisenseconnexin 43 polynucleotide or oligonucleotide. In yet anotherembodiment, the anti-connexin polynucleotide or oligonucleotide e.g., ananti-connexin 43 polynucleotide or oligonucleotide, or the antisensepolynucleotide or oligonucleotide, e.g., an antisense connexin 43polynucleotide or oligonucleotide, is an oligodeoxynucleotide.

According to the invention, polynucleotides, oligonucleotides, antisensepolynucleotides, antisense oligonucleotides, oligodeoxynucleotides andantisense oligodeoxynucleotides may have unmodified or modified backbonestructures.

According to one aspect, the anti-connexin polynucleotide, e.g.,anti-connexin 43 polynucleotide, may be administered parenterally.Alternatively, according to this aspect, the anti-connexinpolynucleotide, e.g., anti-connexin 43 polynucleotide, may beadministered topically. Also, the anti-connexin polynucleotide, e.g.,anti-connexin 43 polynucleotide, may be implanted or instilled orinjected. The anti-connexin polynucleotide, e.g., anti-connexin 43polynucleotide, may be administered in a sustained- or slow-releaseformulation.

Suitable anti-connexin 43 oligonucleotides may be selected from thegroup consisting of GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC (SEQ. ID.NO:1); GTA ATT GCG GGA GGA ATT GTT TCT GTC (SEQ. ID. NO:2); and GGC AAGAGA CAC CAA AGA CAC TAC CAG CAT (SEQ. ID. NO:3).

According to an alternate aspect, the present invention is directed to amethod to determine the anti-fibrotic activity of an anti-connexinpolynucleotide, e.g., anti-connexin 43 polynucleotide, comprisingcontacting cells at risk of having or having fibrosis with ananti-connexin polynucleotide, e.g., anti-connexin 43 polynucleotide, anddetermining the anti-fibrotic effect of said anti-connexinpolynucleotide, e.g., anti-connexin 43 polynucleotide. According to oneembodiment, the method is carried out in vitro. According to analternate embodiment, the method is carried out in vivo.

Compositions and formulations of the invention useful in treating orpreventing fibrosis and fibrotic diseases, conditions and disorders thatemploy anti-connexin polynucleotides, including connexin antisensepolynucleotides, are described and claimed.

In one aspect, the invention provides a pharmaceutical compositioncomprising one or more anti-connexin polynucleotides (e.g. connexinantisense polynucleotides). Preferably, the pharmaceutical compositionfurther comprises a pharmaceutically acceptable carrier, diluent orexcipient. For example, the inventions include pharmaceuticalcompositions comprising (a) a therapeutically effect amount of apharmaceutically acceptable connexin antisense polynucleotide and (b) apharmaceutically acceptable carrier or diluent.

The invention also includes pharmaceutical compositions useful in intreating or preventing fibrosis and fibrotic diseases, disorders andconditions comprising (a) a therapeutically effective amount of ananti-connexin polynucleotide, and (b) a therapeutically effective amountof one or more therapeutic agents. The invention includes pharmaceuticalcompositions useful in treating or preventing fibrosis and fibroticdiseases, disorders and conditions comprising (a) a therapeuticallyeffective amount of an anti-connexin polynucleotide, and (b) atherapeutically effective amount of one or more and/or agents useful inwound healing. The invention includes pharmaceutical compositions usefulin treating or preventing fibrosis and fibrotic diseases, disorders andconditions comprising (a) a therapeutically effective amount of ananti-connexin polynucleotide, and (b) a therapeutically effective amountof one or more and/or anti-fibrotic agents. Preferably, thepharmaceutical compositions further comprise a pharmaceuticallyacceptable carrier, diluent or excipient.

Pharmaceutical compositions with one or more anti-connexinpolynucleotides, alone or with one or more other anti-fibrotic agents,useful in treating or preventing fibrosis and fibrotic diseases,disorders and conditions are provided for combined, simultaneous,separate sequential or sustained administration. In one embodiment, acomposition comprising one or more anti-connexin polynucleotides isadministered at or about the same time as one ore more therapeuticagents, and/or agents useful for wound healing and/or anti-fibroticagents.

Examples of a connexin antisense polynucleotide include, for example, ananti-connexin oligodeoxynucleotide (ODN), including antisense (includingmodified and unmodified backbone antisense; e.g., a DNA antisensepolynucleotide that binds to a connexin mRNA), RNAi, and siRNApolynucleotides.

Suitable connexin antisense polynucleotides include for example,antisense ODNs against connexin 43 (Cx43), connexin 26 (Cx26), connexin37 (Cx37), connexin 30 (Cx30), connexin 31.1 (Cx31.1) and connexin 32(Cx32). In certain embodiments, suitable compositions include multipleconnexin antisense polynucleotides in combination, including forexample, polynucleotides targeting Cx 43, 26, 30, and 31.1. Preferredconnexin antisense polynucleotides target connexin 43.

Conveniently, the oligodeoxynucleotide to connexin 43 is selected from:GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC (SEQ. ID. NO:1); GTA ATT GCG GCAGGA GGA ATT GTT TCT GTC (SEQ. ID. NO:2); GGC AAG AGA CAC CAA AGA CAC TACCAG CAT (SEQ. ID. NO:3), a polynucleotide having at least about 70percent homology with SEQ. ID. NOS:1, 2, or 3 or a polynucleotide whichhybridizes to connexin 43 mRNA under conditions of medium to highstringency.

Pharmaceutical compositions useful in treating or preventing fibrosisand fibrotic diseases, disorders and conditions are also provided in theform of a combined preparation, for example, as an admixture of one ormore anti-connexin polynucleotides and one or more other agents usefulfor wound healing, e.g., growth factors that are effective in promotingor improving wound healing, such as platelet derived growth factor,epidermal growth factor, fibroblast growth factor (e.g., FGF2), vascularendothelial growth factor, and transforming growth factor 133, and/orcytokines that are effective in promoting or improving wound healing,such as IL-7 and IL-10, and/or other agents that are effective inpromoting or improving wound healing, such as IGF (e.g., IGF-1) andIGFBP (e.g., IGFBP-2).

The term “a combined preparation” includes a “kit of parts” in the sensethat the combination partners as defined above can be dosedindependently or by use of different fixed combinations withdistinguished amounts of the combination partners (a) and (b), i.e.simultaneously, separately or sequentially. The parts of the kit canthen, for example, be administered simultaneously or chronologicallystaggered, that is at different time points and with equal or differenttime intervals for any part of the kit of parts.

In a preferred embodiment, the administration of a combined preparationwill have fewer administration time points and/or increased timeintervals between administrations as a result of such combined use.

In another aspect, the invention includes methods for administering atherapeutically effective amount of one or more pharmaceuticallyacceptable connexin antisense polynucleotides formulated in a delayedrelease preparation, a slow release preparation, an extended releasepreparation, a controlled release preparation, and/or in a repeat actionpreparation to a subject with fibrosis or a fibrotic disease, disorderand condition.

In a further aspect, the invention includes transdermal patches,dressings, pads, wraps, matrices and bandages capable of being adheredor otherwise associated with the skin or other tissue of a subject, saidarticles being capable of delivering a therapeutically effective amountof one or more pharmaceutically acceptable anti-connexinpolynucleotides, e.g., connexin antisense polynucleotides to a patientwith fibrosis or fibrotic disease, disorder or condition.

The invention includes devices useful in treating or preventing fibrosisand fibrotic diseases, disorders and conditions containingtherapeutically effective amounts of one or more pharmaceuticallyacceptable anti-connexin polynucleotides, e.g., connexin antisensepolynucleotides, for example, a rate-controlling membrane enclosing adrug reservoir and a monolithic matrix device. These devices may beemployed for the treatment of subjects in need thereof as disclosedherein. Suitably the dressing or matrix is provided including the formof a solid substrate with an anti-connexin polynucleotide, e.g., aconnexin antisense polynucleotide, either alone or in combination withone or more therapeutic agents and/or agents useful for wound healing,dispersed on or in the solid substrate. In one embodiment thepharmaceutical product of the invention is provided in combination witha dressing or matrix. Preferred anti-connexin polynucleotides andconnexin antisense polynucleotides are anti-connexin 43 polynucleotidesand connexin 43 antisense polynucleotides.

In another aspect, the invention includes an article of manufactureuseful in treating or preventing fibrosis and fibrotic diseases,disorders and conditions comprising a vessel containing atherapeutically effective amount of one or more pharmaceuticallyacceptable anti-connexin polynucleotides, e.g., connexin antisensepolynucleotides, and instructions for use. Such instructions may includeinstructions regarding use for the treatment of a subject having afibrosis or a fibrotic disease, disorder or condition. Preferredanti-connexin polynucleotides and connexin antisense polynucleotides areanti-connexin 43 polynucleotides and connexin 43 antisensepolynucleotides. In one embodiment, the vessel further comprises atherapeutically effective amount of one or more therapeutic agentsand/or agents useful for wound healing. In one embodiment, the articleof manufacture additionally comprises a vessel containing atherapeutically effective amount of one or more therapeutic agentsand/or agents useful for wound healing.

The invention includes an article of manufacture useful in treating orpreventing fibrosis and fibrotic diseases, disorders and conditionscomprising packaging material containing one or more dosage formscontaining one or more pharmaceutically acceptable anti-connexinpolynucleotides, e.g., connexin antisense polynucleotides, wherein thepackaging material has a label that indicates that the dosage form canbe used for a subject having or suspected of having or predisposed toany of the diseases, disorders and/or conditions described or referencedherein, including fibrosis or a fibrotic disease, disorder or condition.Such dosage forms include, for example, topical delivery forms andformulations. Preferred anti-connexin polynucleotides and connexinantisense polynucleotides are anti-connexin 43 polynucleotides andconnexin 43 antisense polynucleotides. In one embodiment, the dosageform further comprises a therapeutically effective amount of one or moretherapeutic agents and/or agents useful for wound healing. In oneembodiment, the dosage form additionally comprises a vessel containing atherapeutically effective amount of one or more therapeutic agentsand/or agents useful for wound healing.

DETAILED DESCRIPTION Definitions

As used herein, a “disorder” is any disorder, disease, or condition thatwould benefit from an agent that reduces or retards fibrosis. Forexample, included are diseases, disorders and conditions characterizedby excess production of fibrous material, including excess production offibrous material within the extracellular matrix. Also included arediseases, disorders and conditions characterized by replacement ofnormal tissue elements by abnormal, non-functional, and/or excessiveaccumulation of matrix-associated components.

As used herein, “subject” refers to any mammal, including humans,domestic and farm animals, and zoo, sports, and pet animals, such asdogs, horses, cats, sheep, pigs, cows, etc. The preferred mammal hereinis a human, including adults, children, and the elderly. A subject mayalso be a bird, including zoo, sports, and pet birds.

As used herein, “preventing” means preventing in whole or in part, orameliorating or controlling, or reducing, retarding or halting theproduction or occurrence of the thing or event to be prevented.

As used herein, a “therapeutically effective amount” or “effectiveamount” in reference to the compounds or compositions of the instantinvention refers to the amount sufficient to induce a desiredbiological, pharmaceutical, or therapeutic result. That result can bealleviation of the signs, symptoms, or causes of a disease or disorderor condition, or any other desired alteration of a biological system. Inthe present invention, the result will involve preventing fibrosis.

As used herein, the term “treating” refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those prone to havingthe disorder or diagnosed with the disorder or those in which thedisorder is to be prevented. Thus, anti-fibrotic applications ofcompounds and compositions and formulations of the inventionadministered prior to the formation of fibrosis or fibrotic tissue arewithin the invention.

As used herein, “simultaneously” is used to mean that the one or moreanti-connexin polynucleotides, alone or in combination with one or moretherapeutic agents and/or agents useful for wound healing areadministered concurrently, whereas the term “in combination” is used tomean the polynucleotides and/or agents are administered, if notsimultaneously or in physical combination, then “sequentially” within atimeframe that they both are available to act therapeutically. Thus,administration “sequentially” may permit one polynucleotide or agent tobe administered within minutes (for example, 1, 2, 3, 4, 5, 10, 15, 20,25, 30) minutes or a matter of hours, days, weeks or months after theother polynucleotide or agent provided that both are concurrentlypresent in therapeutically effective amounts. The time delay betweenadministration of the components will vary depending on the exact natureof the components, the interaction there between, and their respectivehalf-lives.

As used herein, an “anti-connexin polynucleotide” decreases or inhibitsexpression of connexin mRNA and/or protein. Anti-connexinpolynucleotides include, without limitation, antisense compounds such asantisense polynucleotides, other polynucleotides (such aspolynucleotides having siRNA or ribozyme functions). Suitable examplesof an anti-connexin polynucleotide include an antisense polynucleotideto a connexin. Accordingly, suitable anti-connexin polynucleotidesinclude, for example, antisense polynucleotides (e.g., connexin 43antisense polynucleotides) that modulate expression or activity ofconnexins and gap junctions in selected tissues, cells, and subjects.Exemplary anti-connexin polynucleotides are further described herein.

As used herein, “fibrotic” diseases, disorders, or conditions includethose mentioned herein, and further include acute and chronic, clinicalor sub-clinical presentation, in which fibrogenic associated biology orpathology is evident. Fibrotic diseases, disorders, or conditionsinclude diseases, disorders or conditions characterized, in whole or inpart, by the excess production of fibrous material, including excessproduction of fibrotic material within the extracellular matrix, or thereplacement of normal tissue elements by abnormal, non-functional,and/or excessive accumulation of matrix-associated components. Fibroticdiseases, disorders, or conditions include, for example,fibrogenic-related biology or pathology characterized by fibrosis.

Exemplary fibrotic diseases, disorders and conditions include, forexample, scleroderma (including morphea, generalized morphea, or linearscleroderma), kidney fibrosis (including glomerular sclerosis, renaltubulointerstitial fibrosis, progressive renal disease or diabeticnephropathy), cardiac fibrosis (e.g. myocardial fibrosis), pulomanryfibrosis (e.g., glomerulosclerosis pulmonary fibrosis, idiopathicpulmonary fibrosis, silicosis, asbestosis, interstitial lung disease,interstitial fibrotic lung disease, and chemotherapy/radiation inducedpulmonary fibrosis), oral fibrosis, endomyocardial fibrosis, deltoidfibrosis, pancreatitis, inflammatory bowel disease, Crohn's disease,nodular fascilitis, eosinophilic fasciitis, general fibrosis syndromecharacterized by replacement of normal muscle tissue by fibrous tissuein varying degrees, retroperitoneal fibrosis, liver fibrosis, livercirrhosis, chronic renal failure; myelofibrosis (bone marrow fibrosis),drug induced ergotism, glioblastoma in Li-Fraumeni syndrome, sporadicglioblastoma, myleoid leukemia, acute myelogenous leukemia,myelodysplastic syndrome, myeloproferative syndrome, gynecologicalcancer, Kaposi's sarcoma, Hansen's disease, collagenous colitis andacute fibrosis.

Fibrotic diseases, disorders and conditions may also includecontractures. Contractures, including post-operative contractures, referto a permanent or long term reduction of range of motion due to tonicspasm or fibrosis, or to loss of normal tissue compliance, motion orequilibrium (e.g., muscle, tendon, ligament, fascia, synovium, jointcapsule, other connective tissue, or fat). In general, the condition ofcontracture may involve a fibrotic response with inflammatorycomponents, both acute and chronic. Some of which may be associated withsurgery, including a release procedure. Hereditary contractures such asDupytren's contracture, Peyronie's disease, and Ledderhose's disease arealso included.

Fibrosis can be either chronic or acute. Fibrotic conditions includeexcessive amounts of fibrous tissue, including excessive amounts ofextracellular matrix accumulation within a tissue, forming tissue whichcauses dysfunction and, potentially, organ failure. Chronic fibrosisincludes fibrosis of the major organs, most commonly lung, liver, kidneyand/or heart. Acute fibrosis (usually with a sudden and severe onset andof short duration) occurs typically as a common response to variousforms of trauma including injuries, ischemic illness (e.g. cardiacscarring following heart attack), environmental pollutants, alcohol andother types of toxins, acute respiratory distress syndrome, radiationand chemotherapy treatments. All tissues damaged by trauma can becomefibrotic, particularly if the damage is repeated.

Response to injury has been reported to involve coordinated andtemporally regulated patterns of mediators and sequence of cellularevents in tissues subsequent to injury. The initial injury is reportedto trigger coagulation cascade and an acute local inflammatory responsefollowed by mesenchymal cell recruitment, proliferation and matrixsynthesis. Uncontrolled matrix accumulation, often involving aberrantcytokine pathways, can lead to fibrotic conditions or disorders.Progressive fibrosis in vital organs such as the lung, kidney, liver,heart, brain and bone marrow, is both a major cause of illness anddeath.

Anti-Connexin Polynucleotides

Anti-connexin polynucleotides include connexin antisense polynucleotidesas well as polynucleotides which have functionalities that enable themto downregulate or otherwise inhibit connexin expression (for example,by downregulation or inhibition of mRNA transcription or translation).In the case of downregulation, this will have the effect of reducingdirect cell-cell communication by gap junctions, or access to theextracellular space through hemichannels, at the site at which connexinexpression is downregulated.

Suitable anti-connexin polynucleotides include antisenseoligonucleotides (including antisense oligodeoxynucleotides), RNAipolynucleotides and siRNA polynucleotides.

Synthesis of antisense polynucleotides and other anti-connexinpolynucleotides such as RNAi, siRNA, and ribozyme polynucleotides aswell as polynucleotides having modified and mixed backbones is known tothose of skill in the art. See e.g. Stein C. A. and Krieg A. M. (eds),Applied Antisense Oligonucleotide Technology, 1998 (Wiley-Liss).

According to one aspect, the downregulation of connexin expression maybe based generally upon the antisense approach using antisensepolynucleotides (such as DNA or RNA polynucleotides), and moreparticularly upon the use of antisense oligodeoxynucleotides (ODN).These polynucleotides (e.g., ODN) target the connexin protein (s) to bedownregulated. Typically the polynucleotides are single stranded, butmay be double stranded.

The antisense polynucleotide may inhibit transcription and/ortranslation of a connexin. Preferably the polynucleotide is a specificinhibitor of transcription and/or translation from the connexin gene ormRNA, and does not inhibit transcription and/or translation from othergenes or mRNAs. The product may bind to the connexin gene or mRNA either(i) 5′ to the coding sequence, and/or (ii) to the coding sequence,and/or (iii) 3′ to the coding sequence.

The antisense polynucleotide is generally antisense to a connexin mRNA.Such a polynucleotide may be capable of hybridizing to the connexin mRNAand may thus inhibit the expression of connexin by interfering with oneor more aspects of connexin mRNA metabolism including transcription,mRNA processing, mRNA transport from the nucleus, translation or mRNAdegradation. The antisense polynucleotide typically hybridizes to theconnexin mRNA to form a duplex which can cause direct inhibition oftranslation and/or destabilization of the mRNA. Such a duplex may besusceptible to degradation by nucleases.

The antisense polynucleotide may hybridize to all or part of theconnexin mRNA. Typically the antisense polynucleotide hybridizes to theribosome binding region or the coding region of the connexin mRNA. Thepolynucleotide may be complementary to all of or a region of theconnexin mRNA. For example, the polynucleotide may be the exactcomplement of all or a part of connexin mRNA. However, absolutecomplementarity is not required and polynucleotides which havesufficient complementarity to form a duplex having a melting temperatureof greater than about 20° C., 30° C. or 40° C. under physiologicalconditions are particularly suitable for use in the present invention.

Thus the polynucleotide is typically a homologue of a sequencecomplementary to the mRNA. The polynucleotide may be a polynucleotidewhich hybridizes to the connexin mRNA under conditions of medium to highstringency such as 0.03M sodium chloride and 0.03M sodium citrate atfrom about 50° C. to about 60° C.

For certain aspects, suitable polynucleotides are typically from about 6to 40 nucleotides in length. Preferably a polynucleotide may be fromabout 12 to about 35 nucleotides in length, or alternatively from about12 to about 20 nucleotides in length or more preferably from about 18 toabout 32 nucleotides in length. According to an alternative aspect, thepolynucleotide may be at least about 40, for example at least about 60or at least about 80, nucleotides in length and up to about 100, about200, about 300, about 400, about 500, about 1000, about 2000 or about3000 or more nucleotides in length.

The connexin protein or proteins targeted by the polynucleotide will bedependent upon the site at which downregulation is to be effected. Thisreflects the non-uniform make-up of gap junction(s) at different sitesthroughout the body in terms of connexin sub-unit composition. Theconnexin is a connexin that naturally occurs in a human or animal in oneaspect or naturally occurs in the tissue in which connexin expression oractivity is to be decreased. The connexin gene (including codingsequence) generally has homology with the coding sequence of one or moreof the specific connexins mentioned herein, such as homology with theconnexin 43 coding sequence shown in Table 2. The connexin is typicallyan a or B connexin. Preferably the connexin is an a connexin and isexpressed in the tissue to be treated.

Some connexin proteins are however more ubiquitous than others in termsof distribution in tissue. One of the most widespread is connexin 43.Polynucleotides targeted to connexin 43 are particularly suitable foruse in the present invention. In other aspects other connexins aretargeted.

In one preferred aspect, the antisense polynucleotides are targeted tothe mRNA of one connexin protein only. Most preferably, this connexinprotein is connexin 43. In another aspect, connexin protein is connexin26, 30, 31.1, 32, 36, 37, 40, or 45. In other aspects, the connexinprotein is connexin 30.3, 31, 40.1, or 46.6.

It is also contemplated that polynucleotides targeted to separateconnexin proteins be used in combination (for example 1, 2, 3, 4 or moredifferent connexins may be targeted). For example, polynucleotidestargeted to connexin 43, and one or more other members of the connexinfamily (such as connexin 26, 30, 30.3, 31.1, 32, 36, 37, 40, 40.1, 45,and 46.6) can be used in combination.

Alternatively, the antisense polynucleotides may be part of compositionswhich may comprise polynucleotides to more than one connexin protein.Preferably, one of the connexin proteins to which polynucleotides aredirected is connexin 43. Other connexin proteins to whicholigodeoxynucleotides are directed may include, for example, connexins26, 30, 30.3, 31.1, 32, 36, 37, 40, 40.1, 45, and 46.6. Suitableexemplary polynucleotides (and ODNs) directed to various connexins areset forth in Table 1.

Individual antisense polynucleotides may be specific to a particularconnexin, or may target 1, 2, 3 or more different connexins. Specificpolynucleotides will generally target sequences in the connexin gene ormRNA which are not conserved between connexins, whereas non-specificpolynucleotides will target conserved sequences for various connexins.

The polynucleotides for use in the invention may suitably be unmodifiedphosphodiester oligomers. Such oligodeoxynucleotides may vary in length.A 30 mer polynucleotide has been found to be particularly suitable.

Many aspects of the invention are described with reference tooligodeoxynucleotides. However it is understood that other suitablepolynucleotides (such as RNA polynucleotides) may be used in theseaspects.

The antisense polynucleotides may be chemically modified. This mayenhance their resistance to nucleases and may enhance their ability toenter cells. For example, phosphorothioate oligonucleotides may be used.Other deoxynucleotide analogs include methylphosphonates,phosphoramidates, phosphorodithioates, N3′P5′-phosphoramidates andoligoribonucleotide phosphorothioates and their 2′-O-alkyl analogs and2′-O-methylribonucleotide methylphosphonates. Alternatively mixedbackbone oligonucleotides (“MBOs”) may be used. MBOs contain segments ofphosphothioate oligodeoxynucleotides and appropriately placed segmentsof modified oligodeoxy- or oligoribonucleotides. MBOs have segments ofphosphorothioate linkages and other segments of other modifiedoligonucleotides, such as methylphosphonate, which is non-ionic, andvery resistant to nucleases or 2′-O-alkyloligoribonucleotides. Methodsof preparing modified backbone and mixed backbone oligonucleotides areknown in the art.

The precise sequence of the antisense polynucleotide used in theinvention will depend upon the target connexin protein. In oneembodiment, suitable connexin antisense polynucleotides can includepolynucleotides such as oligodeoxynucleotides selected from thefollowing sequences set forth in Table 1:

TABLE 1 5′ GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC 3′ (connexin 43)(SEQ.ID.NO: 1) 5′ GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC 3′(connexin 43) (SEQ.ID.NO: 2) 5′GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT 3′ (connexin 43) (SEQ.ID.NO: 3)5′ TCC TGA GCA ATA CCT AAC GAA CAA ATA 3′ (connexin 26) (SEQ.ID.NO: 4)5′ CAT CTC CTT GGT GCT CAA CC 3′ (connexin 37) (SEQ.ID.NO: 5) 5′CTG AAG TCG ACT TGG CTT GG 3′ (connexin 37) (SEQ.ID.NO: 6) 5′CTC AGA TAG TGG CCA GAA TGC 3′ (connexin 30) (SEQ.ID.NO: 7) 5′TTG TCC AGG TGA CTC CAA GG 3′ (connexin 30) (SEQ.ID.NO: 8) 5′CGT CCG AGC CCA GAA AGA TGA GGT C 3′ (connexin 31.1) (SEQ.ID.NO: 9) 5′AGA GGC GCA CGT GAG ACA C 3′ (connexin 31.1) (SEQ.ID.NO: 10) 5′TGA AGA CAA TGA AGA TGT T 3′ (connexin 31.1) (SEQ.ID.NO: 11) 5′TTT CTT TTC TAT GTG CTG TTG GTG A 3′ (connexin 32) (SEQ.ID.NO: 12)

Suitable polynucleotides for the preparation of the combinedpolynucleotide compositions described herein include for example,polynucleotides to connexin 43 and polynucleotides for connexins 26, 30,31.1, 32 and 37 as described in Table 1 above.

Although the precise sequence of the antisense polynucleotide used inthe invention will depend upon the target connexin protein, for connexin43, antisense polynucleotides having the following sequences have beenfound to be particularly suitable:

(SEQ.ID.NO: 1) GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC; (SEQ.ID.NO: 2)GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC; and (SEQ.ID.NO: 3)GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT.

For example, suitable antisense polynucleotides for connexins 26, 31.1and 32 have the following sequences:

(SEQ.ID.NO: 4) 5′ TCC TGA GCA ATA CCT AAC GAA CAA ATA (connexin 26);(SEQ.ID.NO: 9) 5′ CGT CCG AGC CCA GAA AGA TGA GGT C (connexin 31.1); and(SEQ.ID.NO: 12) 5′ TTT CTT TTC TAT GTG CTG TTG GTG A (connexin 32).

Other connexin antisense polynucleotide sequences useful according tothe methods of the present invention include:

(SEQ.ID.NO: 5) 5′ CAT CTC CTT GGT GCT CAA CC 3′ (connexin 37);(SEQ.ID.NO: 6) 5′ CTG AAG TCG ACT TGG CTT GG 3′ (connexin 37);(SEQ.ID.NO: 7) 5′ CTC AGA TAG TGG CCA GAA TGC 3′ (connexin 30);(SEQ.ID.NO: 8) 5′ TTG TCC AGG TGA CTC CAA GG 3′ (connexin 30);(SEQ.ID.NO: 10) 5′ AGA GGC GCA CGT GAG ACA C 3′ (connexin 31.1); and(SEQ.ID.NO: 11) 5′ TGA AGA CAA TGA AGA TGT T 3′ (connexin 31.1).

Polynucleotides, including ODN's, directed to connexin proteins can beselected in terms of their nucleotide sequence by any convenient, andconventional, approach. For example, the computer programs MacVector andOligoTech (from Oligos etc. Eugene, Oreg., USA) can be used. Onceselected, the ODN's can be synthesized using a DNA synthesizer.

Polynucleotide Homologues

Anti-connexin polynucleotides also include polynucleotide homologues.Homology and homologues are discussed herein (for example, thepolynucleotide may be a homologue of a complement to a sequence inconnexin mRNA). Such a polynucleotide typically has at least about 70%homology, preferably at least about 80%, at least about 90%, at leastabout 95%, at least about 97% or at least about 99% homology with therelevant sequence, for example over a region of at least about 15, atleast about 20, at least about 40, at least about 100 more contiguousnucleotides (of the homologous sequence).

Homology may be calculated based on any method in the art. For examplethe UWGCG Package provides the BESTFIT program, which can be used tocalculate homology (for example used on its default settings) (Devereuxet al. (1984) Nucleic Acids Research 12, p 387-395). The PILEUP andBLAST algorithms can be used to calculate homology or line up sequences(typically on their default settings), for example as described inAltschul S. F. (1993) J Mol Evol 36: 290-300; Altschul, S, F et al(1990) J Mol Biol 215: 403-10.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score falls off by the quantity X fromits maximum achieved value; the cumulative score goes to zero or below,due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached.

The BLAST algorithm parameters W, T and X determine the sensitivity andspeed of the alignment. The BLAST program uses as defaults a word length(W), the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to a second sequence isless than about 1, preferably less than about 0.1, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The homologous sequence typically differs from the relevant sequence byat least about (or by no more than about) 2, 5, 10, 15, 20 moremutations (which may be substitutions, deletions or insertions). Thesemutations may be measured across any of the regions mentioned above inrelation to calculating homology.

The homologous sequence typically hybridizes selectively to the originalsequence at a level significantly above background. Selectivehybridization is typically achieved using conditions of medium to highstringency (for example 0.03M sodium chloride and 0.03M sodium citrateat from about 50° C. to about 60° C.). However, such hybridization maybe carried out under any suitable conditions known in the art (seeSambrook et al. (1989), Molecular Cloning: A Laboratory Manual). Forexample, if high stringency is required, suitable conditions include0.2×SSC at 60° C. If lower stringency is required, suitable conditionsinclude 2×SSC at 60° C.

Therapeutic Agents

Therapeutic agents include pharmaceutically acceptable agents useful inthe treatment of wounds or the promotion of wound-healing, whethercurrently existing and known or later developed. Therapeutic agentsinclude, for example, anti-infectives, anesthetics, analgesics,antibiotics, narcotics, and steroidal and non-steroidalanti-inflammatory agents. Preferred therapeutic agents include topicalsteroid anti-inflammatory agents, antimicrobial agents, local andtopical anesthetics, and topical opioids. In certain embodiments, one,two three, four, five or six therapeutic agents may be used incombination. In other embodiments, the therapeutic agent is not ananti-connexin peptide, e.g., an anti-connexin peptidomimetic.

Agents Useful for Wound Healing

As used herein, agents useful for wound healing include stimulators,enhancers or positive mediators of the wound healing cascade which 1)promote or accelerate the natural wound healing process or 2) reduceeffects associated with improper or delayed wound healing, which effectsinclude, for example, adverse inflammation, epithelialization,angiogenesis and matrix deposition, and scarring and fibrosis. Incertain embodiments, the therapeutic agent is not an anti-connexinpeptide, e.g., an anti-connexin peptidomimetic.

Positive mediators, enhancers and stimulators include for example, anagent which may stimulate, enhance, facilitate, or accelerate (i.e.,agonize) the quantity, quality or efficacy of wound healing or theactive wound healing process, or a wound healing-associated growthfactor or cytokine at a wound site, or the activation of a woundhealing-associated growth factor or cytokine receptor. Such agents mayinclude a wound healing-associated growth factor or cytokine or apartially modified form of a wound healing-associated growth factor orcytokine, for example. A partially modified form of woundhealing-associated growth factor or cytokine may, for example, have alonger half-life than the natural wound healing-associated growth factoror cytokine. Alternatively, it may be an inhibitor of woundhealing-associated growth factor or cytokine metabolism.

Agents useful for wound healing also include anti-fibrotic agents, whichinclude, for example, any agent which can prevent and/or suppress,reduce or improve fibrogenic pathology. Exemplary anti-fibrotic agentsinclude, for example, direct or indirect regulators associated with thewound-associated inflammatory reaction, recruitment of neutrophils tothe site of injury; activation and recruitment of macrophages andendothelial cells; recruitment and activation of lymphocytes and/oreosinophils via secretion of a number of cytokines/chemokines; releaseof cytotoxic mediators and fibrogenic cytokines; recruiting andactivating cell proliferation, ECM synthesis and angiogenesis.

Partial modification of such an agent may be by way of addition,deletion or substitution of amino acid residues. A substitution may forexample be a conserved substitution. Hence a partially modified moleculemay be a homologue of the molecule from which it was derived. It mayhave at least about 40%, for example about 50, 60, 70, 80, 90 or 95%,homology with the molecule from which it is derived.

As used herein, agents useful for wound healing may include for example,wound-healing-promoting or scar-reducing agents for wound treatmentmodalities now known in the art or later-developed; exemplary factors,agents or modalities including natural or synthetic growth factors,cytokines, or modulators thereof to promote wound healing, wound healingpromoting bioengineered matrix, dressings bandages, and the like.Suitable examples may include, but not limited to 1) topical or dressingand related therapies and debriding agents (such as, for example,Santyl® collagenase) and Iodosorb® (cadexomer iodine); 2) antimicrobialagents, including systemic or topical creams or gels, including, forexample, silver-containing agents such as SAGs (silver antimicrobialgels), (CollaGUARD™, Innocoll, Inc) (purified type-I collagen proteinbased dressing), CollaGUARD Ag (a collagen-based bioactive dressingimpregnated with silver for infected wounds or wounds at risk ofinfection), DermaSIL™ (a collagen-synthetic foam composite dressing fordeep and heavily exuding wounds); 3) cell therapy or bioengineered skin,skin substitutes, and skin equivalents, including, for example,Dermograft (3-dimensional matrix cultivation of human fibroblasts thatsecrete cytokines and growth factors), Apligraf® (human keratinocytesand fibroblasts), Graftskin® (bilayer of epidermal cells and fibroblaststhat is histologically similar to normal skin and produces growthfactors similar to those produced by normal skin), TransCyte (a HumanFibroblast Derived Temporary Skin Substitute) and Oasis® (an activebiomaterial that comprises both growth factors and extracellular matrixcomponents such as collagen, proteoglycans, and glycosaminoglycans); 4)cytokines, growth factors or hormones (both natural and synthetic)introduced to the wound to promote wound healing, including, forexample, NGF, NT3, BDGF, integrins, plasmin, semaphoring, blood-derivedgrowth factor, keratinocyte growth factor, tissue growth factor,TGF-alpha, TGF-beta, PDGF (one or more of the three subtypes may beused: AA, AB, and B), PDGF-BB, TGF-beta 3, factors that modulate therelative levels of TGFβ3, TGFβ1, and TGFβ2 (e.g., Mannose-6-phosphate),sex steroids, including for example, estrogen, estradiol, or anoestrogen receptor agonist selected from the group consisting ofethinyloestradiol, dienoestrol, mestranol, oestradiol, oestriol, aconjugated oestrogen, piperazine oestrone sulphate, stilboestrol,fosfesterol tetrasodium, polyestradiol phosphate, tibolone, aphytoestrogen, 17-beta-estradiol; thymic hormones such asThymosin-beta-4, EGF, HB-EGF, fibroblast growth factors (e.g., FGF1,FGF2, FGF7), keratinocyte growth factor, TNF, interleukins family ofinflammatory response modulators such as, for example, IL-10, IL-1,IL-2, IL-6, IL-8, and IL-10 and modulators thereof; INFs (INF-alpha,-beta, and -delta); stimulators of activin or inhibin, and inhibitors ofinterferon gamma prostaglandin E2 (PGE2) and of mediators of theadenosine 3′,5′-cyclic monophosphate (cAMP) pathway; adenosine A1agonist, adenosine A2 agonist or 5) other agents useful for woundhealing, including, for example, both natural or synthetic homologues,agonist and antagonist of VEGF, VEGFA, IGF; IGF-1, proinflammatorycytokines, GM-CSF, and leptins and 6) IGF-1 and KGF cDNA, autologousplatelet gel, hypochlorous acid (Sterilox® lipoic acid, nitric oxidesynthase3, matrix metalloproteinase 9 (MMP-9), CCT-ETA, alphavbeta6integrin, growth factor-primed fibroblasts and Decorin, silvercontaining wound dressings, Xenaderm™, papain wound debriding agents,lactoferrin, substance P, collagen, and silver-ORC, placental alkalinephosphatase or placental growth factor, modulators of hedgehogsignaling, modulators of cholesterol synthesis pathway, and APC(Activated Protein C), keratinocyte growth factor, TNF, Thromboxane A2,NGF, BMP bone morphogenetic protein, CTGF (connective tissue growthfactor), wound healing chemokines, decorin, modulators of lactateinduced neovascularization, cod liver oil, placental alkalinephosphatase or placental growth factor, and thymosin beta 4. In certainembodiments, one, two three, four, five or six agents useful for woundhealing may be used in combination.

It is to be understood that the agents useful for wound healing(including for example, growth factors and cytokines) above encompassall naturally occurring polymorphs (for example, polymorphs of thegrowth factors or cytokines). Also, functional fragments, chimericproteins comprising one of said agents useful for wound healing or afunctional fragment thereof, homologues obtained by analogoussubstitution of one or more amino acids of the agent useful for woundhealing, and species homologues are encompassed. It is contemplated thatone or more agents useful for wound healing may be a product ofrecombinant DNA technology, and one or more agents useful for woundhealing may be a product of transgenic technology. For example, plateletderived growth factor may be provided in the form of a recombinant PDGFor a gene therapy vector comprising a coding sequence for PDGF.

A fragment or partially modified form thereof refers to a fragment orpartially modified form of the agent useful for wound healing whichretains the biological or wound healing functionality of the factor,although it may of course have additional functionality. Partialmodification may, for example, be by way of addition, deletion orsubstitution of amino acid residues. For example, a substitution may bea conserved substitution. Hence the partially modified molecules may behomologues of the agent useful for wound healing. They may, for example,have at least about 40% homology with said factor. They may for examplehave at least about 50, 60, 70, 80, 90 or 95% homology with said factor.For example, in certain embodiments, IL-10 or a fragment or a partiallymodified form thereof may be administered at a concentration of betweenabout 1 μM and about 10 μM. It may be administered at a concentration ofbetween about 2.5 μM and about 5 μM. In certain other embodiments, IL-10or a fragment or a partially modified form thereof may be administeredimmediately prior to wound healing, but may be effective if administeredwithin about 7 days of wounding. It could be administered on at leasttwo occasions.

Dosage Forms and Formulations and Administration

The agents of the invention of the may be administered to a subject inneed of treatment, such as a subject with, or at risk for, any of thediseases, disorders or conditions mentioned herein. The condition of thesubject can thus be improved. The anti-connexin polynucleotide may beused in the treatment of the subject's body by therapy. They may be usedin the manufacture of a medicament to treat any of the diseases,disorders or conditions mentioned herein.

The anti-connexin polynucleotide may be present in a substantiallyisolated form. It will be understood that the product may be mixed withcarriers or diluents which will not interfere with the intended purposeof the product and still be regarded as substantially isolated. Aproduct of the invention may also be in a substantially purified form,in which case it will generally comprise about 80%, 85%, or 90%,including, for example, at least about 95%, at least about 98% or atleast about 99% of the polynucleotide or dry mass of the preparation.

Depending on the intended route of administration, the pharmaceuticalproducts, pharmaceutical compositions, combined preparations andmedicaments of the invention may, for example, take the form ofsolutions, suspensions, instillations, sprays, salves, creams, gels,foams, ointments, emulsions, lotions, paints, sustained releaseformulations, or powders, and typically contain about 0.01% to about 1%of active ingredient(s), about 1%-50% or active ingredient(s), about2%-60% of active ingredient(s), about 2%-70% of active ingredient(s), orup to about 90% of active ingredient(s). Other suitable formulationsinclude pluronic gel-based formulations, carboxymethylcellulose(CMC)-based formulations, and hyroxypropylmethylcellulose (HPMC)-basedformulations. Other useful formulations include slow or delayed releasepreparations.

Gels or jellies may be produced using a suitable gelling agentincluding, but not limited to, gelatin, tragacanth, or a cellulosederivative and may include glycerol as a humectant, emollient, andpreservative. Ointments are semi-solid preparations that consist of theactive ingredient incorporated into a fatty, waxy, or synthetic base.Examples of suitable creams include, but are not limited to,water-in-oil and oil-in-water emulsions. Water-in-oil creams may beformulated by using a suitable emulsifying agent with propertiessimilar, but not limited, to those of the fatty alcohols such as cetylalcohol or cetostearyl alcohol and to emulsifying wax. Oil-in-watercreams may be formulated using an emulsifying agent such as cetomacrogolemulsifying wax. Suitable properties include the ability to modify theviscosity of the emulsion and both physical and chemical stability overa wide range of pH. The water soluble or miscible cream base may containa preservative system and may also be buffered to maintain an acceptablephysiological pH.

Foam preparations may be formulated to be delivered from a pressurizedaerosol canister, via a suitable applicator, using inert propellants.Suitable excipients for the formulation of the foam base include, butare not limited to, propylene glycol, emulsifying wax, cetyl alcohol,and glyceryl stearate. Potential preservatives include methylparaben andpropylparaben.

Preferably the agents of the invention are combined with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline.Suitable diluents and excipients also include, for example, water,saline, dextrose, glycerol, or the like, and combinations thereof. Inaddition, if desired substances such as wetting or emulsifying agents,stabilizing or ph buffering agents may also be present.

The term “pharmaceutically acceptable carrier” refers to anypharmaceutical carrier that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich can be administered without undue toxicity. Suitable carriers canbe large, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, and amino acid copolymers.

Pharmaceutically acceptable salts can also be present, e.g., mineralacid salts such as hydrochlorides, hydrobromides, phosphates, sulfates,and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like.

Suitable carrier materials include any carrier or vehicle commonly usedas a base for creams, lotions, sprays, foams, gels, emulsions, lotionsor paints for topical administration. Examples include emulsifyingagents, inert carriers including hydrocarbon bases, emulsifying bases,non-toxic solvents or water-soluble bases. Particularly suitableexamples include pluronics, HPMC, CMC and other cellulose-basedingredients, lanolin, hard paraffin, liquid paraffin, soft yellowparaffin or soft white paraffin, white beeswax, yellow beeswax,cetostearyl alcohol, cetyl alcohol, dimethicones, emulsifying waxes,isopropyl myristate, microcrystalline wax, oleyl alcohol and stearylalcohol.

Preferably, the pharmaceutically acceptable carrier or vehicle is a gel,suitably a nonionic polyoxyethylene-polyoxypropylene copolymer gel, forexample, a Pluronic gel, preferably Pluronic F-127 (BASF Corp.). Thisgel is particularly preferred as it is a liquid at low temperatures butrapidly sets at physiological temperatures, which confines the releaseof the agent to the site of application or immediately adjacent thatsite.

An auxiliary agent such as casein, gelatin, albumin, glue, sodiumalginate, carboxymethylcellulose, methylcellulose, hydroxyethylcelluloseor polyvinyl alcohol may also be included in the formulation of theinvention.

Other suitable formulations include pluronic gel-based formulations,carboxymethylcellulose (CMC)-based formulations, andhydroxypropylmethylcellulose (HPMC)-based formulations. The compositionmay be formulated for any desired form of delivery, including topical,instillation, parenteral, intramuscular, subcutaneous, or transdermaladministration. Other useful formulations include slow or delayedrelease preparations.

The formulation which is administered may contain transfection agents.Examples of such agents include cationic agents (for example calciumphosphate and DEAE-dextran) and lipofectants (for example Lipofectam™and Transfectam™), and surfactants.

In one embodiment, the formulation further includes a surfactant toassist with polynucleotide cell penetration or the formulation maycontain any suitable loading agent. Any suitable non-toxic surfactantmay be included, such as DMSO. Alternatively a transdermal penetrationagent such as urea may be included.

Optionally, the anti-connexin polynucleotide may be formulated with oneor more therapeutic agents, agents useful for wound healing, and/oranti-fibrotic agents. In certain embodiments, one, two three, four, fiveor six therapeutic agents may be used in combination. In certainembodiments, one, two three, four, five or six agents useful for woundhealing may be used in combination.

In one aspect, the one or more anti-connexin polynucleotides, eitheralone or in combination with one or more therapeutic agents and/oragents useful in wound healing are provided in the form of a wounddressing or matrix. In certain embodiments, the one or moreanti-connexin polynucleotides (with or without one or more therapeuticagents or agents useful in wound healing) are provided in the form of aliquid, semi solid or solid composition for application directly, or thecomposition is applied to the surface of, or incorporated into, a solidcontacting layer such as a dressing gauze or matrix. The wound dressingcomposition may be provided for example, in the form of a fluid or agel. The one or more anti-connexin polynucleotides (with or without oneor more therapeutic agents or agents useful in wound healing) may beprovided in combination with conventional pharmaceutical excipients fortopical application. Suitable carriers include: Pluronic gels, Polaxamergels, Hydrogels containing cellulose derivatives, including hydroxyethylcellulose, hydroxymethyl cellulose, carboxymethyl cellulose,hydroxypropylmethyl cellulose and mixtures thereof; and hydrogelscontaining polyacrylic acid (Carbopols). Suitable carriers also includecreams/ointments used for topical pharmaceutical preparations, e.g.,creams based on cetomacrogol emulsifying ointment. The above carriersmay include alginate (as a thickener or stimulant), preservatives suchas benzyl alcohol, buffers to control pH such as disodium hydrogenphosphate/sodium dihydrogen phosphate, agents to adjust osmolarity suchas sodium chloride, and stabilizers such as EDTA.

The effective dose for a given subject preferably lies within the dosethat is therapeutically effective for at least 50% of the population,and that exhibits little or no toxicity at this level.

The effective dosage of each of the anti-connexin polynucleotidesemployed in the methods and compositions of the invention may varydepending on a number of factors including the particular anti-connexinpolynucleotide employed, the mode of administration, the frequency ofadministration, the wound being treated, the severity of the wound beingtreated, the route of administration, the needs of a patientsub-population to be treated or the needs of the individual patientwhich different needs can be due to age, sex, body weight, relevantmedical wound specific to the patient.

A suitable dose may be from about 0.001 to about 1 mg/kg body weightsuch as about 0.01 to about 0.4 mg/kg body weight. A suitable dose mayhowever be from about 0.001 to about 0.1 mg/kg body weight such as about0.01 to about 0.050 mg/kg body weight. Doses from about 1 to 100,100-200, 200-300, 300-400, and 400-500 micrograms or more and up toabout 500-1000 micrograms are appropriate. As noted herein, repeatapplications are contemplated. Repeat applications are typically appliedabout once per week, or when healing may appear to be stalled orslowing.

Still other dosage levels between about 1 nanogram (ng)/kg and about 1mg/kg body weight per day of each of the agents described herein. Incertain embodiments, the dosage of each of the subject compounds willgenerally be in the range of about 1 ng to about 1 microgram per kg bodyweight, about 1 ng to about 0.1 microgram per kg body weight, about 1 ngto about 10 ng per kg body weight, about 10 ng to about 0.1 microgramper kg body weight, about 0.1 microgram to about 1 microgram per kg bodyweight, about 20 ng to about 100 ng per kg body weight, about 0.001 mgto about 100 mg per kg body weight, about 0.01 mg to about 10 mg per kgbody weight, or about 0.1 mg to about 1 mg per kg body weight. Incertain embodiments, the dosage of each of the subject compounds willgenerally be in the range of about 0.001 mg to about 0.01 mg per kg bodyweight, about 0.01 mg to about 0.1 mg per kg body weight, about 0.1 mgto about 1 mg per kg body weight, or about 1 mg per kg body weight. Ifmore than one anti-connexin polynucleotide is used, the dosage of eachanti-connexin polynucleotide need not be in the same range as the other.For example, the dosage of one anti-connexin polynucleotide may bebetween about 0.01 mg to about 1 mg per kg body weight, and the dosageof another anti-connexin polynucleotide may be between about 0.1 mg toabout 1 mg per kg body weight. As noted herein, repeat applications arecontemplated. Repeat applications are typically applied about once perweek, or when wound-healing may appear to be stalled or slowing.

Other useful doses range from about 1 to about 10 micrograms per squarecentimeter of the size of the wound or the area to be treated. Certaindoses will be about 1-2, about 1-5, about 2-4, about 5-7, and about 8-10micrograms per square centimeter of the size of the wound or the area tobe treated. Other useful doses are greater than about 10 micrograms persquare centimeter of the size of the wound or the area to be treated,including about 15 micrograms per square centimeter of the size of thewound or the area to be treated about 20 micrograms per squarecentimeter of the size of the wound or the area to be treated about 25micrograms per square centimeter of the size of the wound or the area tobe treated about 30 micrograms per square centimeter of the size of thewound or the area to be treated about 35 micrograms per squarecentimeter of the size of the wound or the area to be treated about 40micrograms per square centimeter of the size of the wound or the area tobe treated, about 50 micrograms per square centimeter of the size of thewound or the area to be treated, and about 100 micrograms per squarecentimeter of the size of the wound or the area to be treated. Otheruseful doses are about 150 micrograms per square centimeter of the sizeof the wound or the area to be treated, about 200 micrograms per squarecentimeter of the size of the wound or the area to be treated, about 250micrograms per square centimeter of wound size, or about 500 microgramsper square centimeter of the size of the wound or the area to betreated. As noted herein, repeat applications are contemplated. Repeatapplications are typically applied about once per week, or whenwound-healing may appear to be stalled or slowing.

For example, in certain embodiments, the anti-connexin polynucleotidecomposition may be applied at about 0.01 micromolar (μM) or 0.05 μM toabout 200 μM final concentration at the treatment site and/or adjacentto the treatment site. Preferably, the antisense polynucleotidecomposition is applied at about 0.05 μM to about 100 μM finalconcentration, more preferably, the anti-connexin polynucleotidecomposition is applied at about 1.0 μM to about 50 μM finalconcentration, and more preferably, the anti-connexin polynucleotidecomposition is applied at about 5-10 μM to about 30-50 μM finalconcentration. Additionally, the anti-connexin polynucleotidecomposition is applied at about 8 μM to about 20 μM final concentration,and alternatively the anti-connexin polynucleotide composition isapplied at about 10 μM to about 20 μM final concentration, or at about10 to about 15 μM final concentration. In certain other embodiments, theanti-connexin polynucleotide is applied at about 10 μM finalconcentration. In yet another embodiment, the anti-connexinpolynucleotide composition is applied at about 1-15 μM finalconcentration. The dose at which an anti-connexin agent is administeredto a patient will depend upon a variety of factors such as the age,weight and general condition of the patient, the condition that is beingtreated, and the particular anti-connexin agent that is beingadministered.

A suitable therapeutically effective dose of an anti-connexin agent maybe from about 0.001 to about 1 mg/kg body weight such as about 0.01 toabout 0.4 mg/kg body weight. A suitable dose may however be from about0.001 to about 0.1 mg/kg body weight such as about 0.01 to about 0.050mg/kg body weight.

Therapeutically effective doses of anti-connexin agents from about 1 to100, 100-200, 100- or 200-300, 100- or 200- or 300-400, and 100- or 200-or 300- or 400-500 micrograms are appropriate. Doses from about 1-1000micrograms are also appropriate. Doses up to 2 milligrams may also beused. Doses are adjusted appropriately when the anti-connexin agent oragents are provided in the form of a dressing, typically upward tomaintain the desired total dose administration.

Alternatively, in the case of anti-connexin oligonucleotides, the dosageof each of the agents in the compositions may be determined by referenceto the composition's concentration relative to the size, length, depth,area or volume of the area to which it will be applied. For example, incertain topical applications, dosing of the pharmaceutical compositionsmay be calculated based on mass (e.g. grams) of or the concentration ina pharmaceutical composition (e.g. μg/ul) per length, depth, area, orvolume of the area of application. Useful doses range from about 1 toabout 10 micrograms per square centimeter of wound size. Certain doseswill be about 1-2, about 1-5, about 2-4, about 5-7, and about 8-10micrograms per square centimeter of wound size. Other useful doses aregreater than about 10 micrograms per square centimeter of wound size,including at least about 15 micrograms per square centimeter of woundsize, at least about 20 micrograms per square centimeter of wound size,at least about 25 micrograms per square centimeter of wound size, about30 micrograms per square centimeter of wound size, at least about 35micrograms per square centimeter of wound size, at least about 40micrograms per square centimeter of wound size, at least about 50micrograms per square centimeter of wound size, and at least about 100to at least about 150 micrograms per square centimeter of wound size.Other doses include about 150-200 micrograms per square centimeter,about 200-250 micrograms per square centimeter, about 250-300 microgramsper square centimeter, about 300-350 micrograms per square centimeter,about 350-400 micrograms per square centimeter, and about 400-500micrograms per square centimeter.

In certain embodiments, the anti-connexin polynucleotide composition maybe applied at about 0.01 micromolar (μM) or 0.05 μM to about 200 μM, orup to 300 μM or up to 1000 μM or up to 2000 μM or up to 3200 μM or morefinal concentration at the treatment site and/or adjacent to thetreatment site, and any doses and dose ranges within these dose numbers.Preferably, the antisense polynucleotide composition is applied at about0.05 μM to about 100 μM final concentration, more preferably, theanti-connexin polynucleotide composition is applied at about 1.0 μM toabout 50 μM final concentration, and more preferably, the anti-connexinpolynucleotide composition is applied at about 5-10 μM to about 30-50 μMfinal concentration. Additionally, the combined anti-connexinpolynucleotide composition is applied at about 8 μM to about 20 μM finalconcentration, and alternatively the anti-connexin polynucleotidecomposition is applied at about 10 μM to about 20 μM finalconcentration, or at about 10 to about 15 μM final concentration. Incertain other embodiments, the anti-connexin polynucleotide is appliedat about 10 μM final concentration. In yet another embodiment, theanti-connexin polynucleotide composition is applied at about 1-15 μMfinal concentration. In other embodiments, the anti-connexinpolynucleotide is applied at about a 20 μM, 30 μM, 40 μM, 50 μM, 60 μM,70 μM, 80 μM, 90 μM, 100 μM, 10-200 μM, 200-300 μM, 300-400 μM, 400-500μM, 500-600 μM, 600-700 μM, 700-800 μM, 800-900 μM, 900-1000 or1000-1500 μM, or 1500 μM-2000 μM or 2000 μM-3000 μM or greater.

Anti-connexin polynucleotide dose amounts include, for example, about0.1-1, 1-2, 2-3, 3-4, or 4-5 micrograms (μg), from about 5 to about 10μg, from about 10 to about 15 μg, from about 15 to about 20 μg, fromabout 20 to about 30 μg, from about 30 to about 40 μg, from about 40 toabout 50 μg, from about 50 to about 75 μg, from about 75 to about 100μg, from about 100 μg to about 250 μg, and from 250 μg to about 500 μg.Dose amounts from 0.5 to about 1.0 milligrams or more or also provided,as noted above. Dose volumes will depend on the size of the site to betreated, and may range, for example, from about 25-100 μL to about100-200 μL, from about 200-500 μL to about 500-1000 pt. Milliliter dosesare also appropriate for larger treatment sites. As noted herein, repeatapplications are contemplated. Repeat applications are typically appliedabout once per week, or when wound-healing may appear to be stalled orslowing.

Conveniently, the anti-connexin polynucleotide is administered in asufficient amount to downregulate expression of a connexin protein, ormodulate gap junction formation for at least about 0.5 to 1 hour, atleast about 1-2 hours, at least about 2-4 hours, at least about 4-6hours, at least about 6-8 hours, at least about 8-10 hours, at leastabout 12 hours, or at least about 24 hours post-administration.

The dosage of each of the anti-connexin polynucleotides in thecompositions and methods of the subject invention may also be determinedby reference to the concentration of the composition relative to thesize, length, depth, area or volume of the area to which it will beapplied. For example, in certain topical and other applications, e.g.,instillation, dosing of the pharmaceutical compositions may becalculated based on mass (e.g. micrograms) of or the concentration in apharmaceutical composition (e.g. μg/μl) per length, depth, area, orvolume of the area of application.

The initial and any subsequent dosages administered will depend uponfactors noted herein. Depending on the oligonucelotide, the dosage andprotocol for administration will vary, and the dosage will also dependon the method of administration selected, for example, local or topicaladministration.

The doses may be administered in single or divided applications. Thedoses may be administered once, or application may be repeated.Typically, application will be repeated weekly until healing ispromoted, or a repeat application may be made in the event that healingslows or is stalled. Doses may be applied 3-7 days apart, or more.Repeat applications may be made, for example, weekly, or bi-weekly, ormonthly or in other frequency for example if and when wound healingslows or is stalled. For some indications, such as certain ocular uses,more frequent dosing, up to hourly may employed.

Agents useful for wound healing suitable for the preparation of thepharmaceutical compositions described herein may be prepared andadministered using methods as known in the art (see, for example, U.S.Pat. Nos. 7,098,190, 6,319,907, 6,331,298, 6,387,364, 6,455,569,6,566,339, 6,696,433, 6,855,505, 6,900,181, 7,052,684 and EP1100529 B1.The concentration of each anti-connexin polynucleotide and agent usefulfor wound healing need not be in the same range as the other. Otheramounts will be known to those of skill in the art and readilydetermined. For example, suitable combination dosages and formulationsin accordance with various aspects and embodiments as described hereinmay be administered according to the dosing regimen as described in U.S.Pat. No. 6,903,078 to Lewis entitled “Combination PDGF, KGF, IGF, andIGFBP for wound healing.”

The initial and any subsequent dosages administered will depend upon thepatient's age, weight, condition, and the disease, wound, disorder orbiological condition being treated. Depending on the agent useful forwound healing, the dosage and protocol for administration will vary, andthe dosage will also depend on the method of administration selected,for example, local or systemic administration.

The agent useful for wound healing may be applied internally orexternally, and may be directed towards any tissue exhibiting a fibroticlesion or area, or at risk thereof. For topical administration of IGF,for example, a zinc oxide formulation can be applied, which induces thelocal production of IGF, as described in Tarnow et al, Scand J. PlastReconstr Hand Surg. 28: 255-259 (1994). An effective dose of PDGF hasbeen reported to be 5 ng/mm² or higher when applied topically asdescribed in U.S. Pat. No. 4,861,757, and at least 1 ng/ml localconcentration of an isoform of PDGF (for example, PDGF-AA, PDGF-BB, orPDGF-AB), up to about 30 ng/ml local concentration applied to apopulation of fibroblasts as described in Lepisto et al., BiochemBiophys Res. Comm 209: 393-399 (1995). PDGF can be administered in acarboxymethylcellulose gel formulation at concentrations of about 10μg/gm to about 500 μg/gm of gel, about 20 μg/gm to about 200 μg/gm, andabout 30 μg/gm to about 100 μg/gm of gel, optimally about 100 μg/gm ofgel. Efficacy of PDGF has been achieved within the range of about 3μg/ml solution to about 300 μg/ml of solution administered.

About 50 μl of KGF of a concentration of about 5 μg/ml may be effectivefor wound healing by topical application to epithelial tissue asdescribed in Sotozono et al, Invest. Opthal. Vis. Science 36: 1524-29(1995). As described in U.S. Pat. No. 4,861,757, an effective amount ofIGF when co-administered with PDGF is in the range of at least 2.5ng/mm² to about 5 ng/mm², with a ratio of PDGF to IGF in the range ofabout 1:10 to about 25:1 weight to weight, with the most effectiveratios being PDGF to IGF of about 1:1 to about 2:1 weight to weight.IGFBP administered in combination with IGF has been shown to increasewound healing at dose levels of about 5 μg of IGF with about 1.5 μg ofphosphorylated IGFBP in a molar ration of about 11:1 IGF:IGFBP, asdescribed in Jyung et al, Surgery 115:233-239 (1994).

For administration of polypeptide therapeutics, for example, PDGF, KGF,IGF and IGFBP polypeptides, the dosage can be in the range of about 5 μgto about 50 μg/kg of tissue to which the application is directed, alsoabout 50 μg to about 5 mg/kg, also about 100 μg to about 500 μg/kg oftissue, and about 200 to about 250 μg/kg. For polynucleotidetherapeutics, for example in a gene therapy administration protocol,depending on the expression strength the polynucleotide in the patient,for tissue targeted administration, vectors containing expressibleconstructs including PDGF, KGF, IGF, and IGFBP coding sequences can beadministered in a range of about 100 ng to about 200 mg of DNA for localadministration in a gene therapy protocol, also about 500 ng to about 50mg, also about 1 μg to about 2 mg of DNA, about 5 μg of DNA to about 500μg of DNA, and about 20 μg to about 100 μg during a local administrationin a gene therapy protocol, and about 250 μg, per injection oradministration. Factors such as method of action and efficacy oftransformation and expression are therefore considerations that willeffect the dosage required for ultimate efficacy for administration ofDNA therapeutics. Where greater expression is desired, over a largerarea of tissue, larger amounts of DNA or the same amountsre-administered in a successive protocol of administrations, or severaladministrations to different adjacent or close tissue portions of forexample, a wound site may be required to effect a positive therapeuticoutcome.

Therapeutic agents suitable for the preparation of the pharmaceuticalcompositions described herein may be formulated and administered usingmethods as known in the art. The initial and any subsequent dosagesadministered will depend upon the patient's age, weight, condition, andthe disease, wound, disorder or biological condition being treated.Depending on the therapeutic, the dosage and protocol for administrationwill vary, and the dosage will also depend on the method ofadministration selected, for example, local or systemic administration.

As noted herein, the doses of either an anti-connexin polynucleotides oranother agent administered in combination can be adjusted down from thedoses administered when given alone.

The combined use of several agents may reduce the required dosage forany individual agent because the onset and duration of effect of thedifferent agents may be complementary. In a preferred embodiment, thecombined use of one or more anti-connexin polynucleotides and one ormore therapeutic agents, agents useful for wound healing, and/or gapjunction modifying agents has an additive, synergistic or super-additiveeffect.

In some cases, the combination of one or more anti-connexinpolynucleotides and one or more therapeutic agents and/or one or moreagents useful for wound healing have an additive effect. In other cases,the combination can have greater-than-additive effect. Such an effect isreferred to herein as a “supra-additive” effect, and may be due tosynergistic or potentiated interaction.

The term “supra-additive promotion of wound healing” refers to a meanwound healing produced by administration of a combination of ananti-connexin polynucleotide and one or more therapeutic agents and/oragents useful for wound healing, is statistically significantly higherthan the sum of the wound healing produced by the individualadministration of either any of the agents alone. Whether produced bycombination administration of an anti-connexin polynucleotide and one ormore therapeutic agents and/or agents useful for wound healing is“statistically significantly higher” than the expected additive value ofthe individual compounds may be determined by a variety of statisticalmethods as described herein and/or known by one of ordinary skill in theart. The term “synergistic” refers to a type of supra-additiveinhibition in which both the anti-connexin polynucleotide and one ormore therapeutic agents, agents useful for wound healing and/or gapjunction modifying agents individually have the ability to promote woundhealing or reduce fibrosis and scarring. The term “potentiated” refersto type of supra-additive effect in which one of the anti-connexinpolynucleotide or one or more therapeutic agents and/or agents usefulfor wound healing individually has the increased ability to promotewound healing.

In general, potentiation may be assessed by determining whether thecombination treatment produces a mean wound healing increase in atreatment group that is statistically significantly supra-additive whencompared to the sum of the mean wound healing increases produced by theindividual treatments in their treatment groups respectively. The meanwound healing increase may be calculated as the difference betweencontrol group and treatment group mean wound healing. The fractionalincrease in wound healing, “fraction affected” (Fa), may be calculatedby dividing the treatment group mean wound healing increase by controlgroup mean wound healing. Testing for statistically significantpotentiation requires the calculation of Fa for each treatment group.The expected additive Fa for a combination treatment may be taken to bethe sum of mean Fas from groups receiving either element of thecombination. The Two-Tailed One-Sample T-Test, for example, may be usedto evaluate how likely it is that the result obtained by the experimentis due to chance alone, as measured by the p-value. A p-value of lessthan 0.05 is considered statistically significant, that is, not likelyto be due to chance alone. Thus, Fa for the combination treatment groupmust be statistically significantly higher than the expected additive Fafor the single element treatment groups to deem the combination asresulting in a potentiated supra-additive effect.

Whether a synergistic effect results from a combination treatment may beevaluated by the median-effect/combination-index isobologram method(Chou, T., and Talalay, P. (1984) Ad. Enzyme Reg. 22:27-55). In thismethod, combination index (CI) values are calculated for differentdose-effect levels based on parameters derived from median-effect plotsof the anti-connexin polynucleotide alone, the one or more agents usefulfor wound healing alone, and the combination of the two at fixed molarratios. CI values of & lt; 1 indicate synergy, CI-1 indicates anadditive effect, and CP1 indicates an antagonistic effect. This analysismay be performed using computer software tools, such as CalcuSyn,Windows Software for Dose Effect Analysis (Biosoft (D, Cambridge UK).

Any method known or later developed in the art for analyzing whether asupra-additive effect exists for a combination therapy is contemplatedfor use in screening for suitable anti-connexin polynucleotides for usein combination with one or more therapeutic agents and/or agents usefulfor wound healing.

In another preferred embodiment, the combined use of one or moreanti-connexin polynucleotides and one or more therapeutic agents and/oragents useful for wound healing reduces the effective dose of any suchagent compared to the effective dose when said agent administered alone.In certain embodiments, the effective dose of the agent when used incombination with one or more anti-connexin polynucleotides is about 1/15to about ½, about 1/10 to about ⅓, about ⅛ to about ⅙, about ⅕, about ¼,about ⅓ or about ½ the dose of the agent when used alone.

In another preferred embodiment, the combined use of one or moreanti-connexin polynucleotides and one or more therapeutic agents and/oragents useful for wound healing reduces the frequency in which saidagent is administered compared to the frequency when said agent isadministered alone. Thus, these combinations allow the use of lowerand/or fewer doses of each agent than previously required to achievedesired therapeutic goals.

The doses may be administered in single or divided applications. Thedoses may be administered once, or application may be repeated.

One or more anti-connexin polynucleotides, either alone or incombination with one or more therapeutic agents and/or one or moreagents useful in wound healing, may be administered by the same ordifferent routes. The various agents of the invention can beadministered separately at different times during the course of therapy,or concurrently in divided or single combination forms.

Preferably one or more anti-connexin polynucleotides useful in thetreatment of fibrosis are delivered by topical administration(peripherally or directly to a site), including but not limited totopical administration using solid supports (such as dressings and othermatrices) and medicinal formulations (such as gels, mixtures,suspensions and ointments). In one embodiment, the solid supportcomprises a biocompatible membrane or insertion into a treatment site.In another embodiment, the solid support comprises a dressing or matrix.In one embodiment of the invention, the solid support composition may bea slow release solid support composition, in which the one or moreanti-connexin polynucleotides useful for wound healing is dispersed in aslow release solid matrix such as a matrix of alginate, collagen, or asynthetic bioabsorbable polymer. Preferably, the solid supportcomposition is sterile or low bio-burden. In one embodiment, a washsolution comprising one or more anti-connexin polynucleotides can beused.

In another embodiment, lavage solution containing about 1 to about 100μg/cm² (preferably about 10 to about 50 μg/cm²) of an anti-connexinagent, would be used at the time of or immediately following injury orsurgery. In all of the embodiments, other anti-connexin polynucleotideswould be administered at equivalent doses adjusted for potency andtolerability of the polynucleotide.

The delivery of one or more anti-connexin polynucleotides (with orwithout one or more therapeutic agents or agents useful for woundhealing) may occur over a period of time, in some instances for about0.5 hours, 1-2 hours, about 2-4 hours, about 4-6 hours, about 6-8, orabout 24 hours or longer, may be a particular advantage in more severewounds. In some instances, cell loss may extend well beyond the site ofa procedure to surrounding cells. Such loss may occur within 24 hours ofthe original procedure and is mediated by gap junction cell-cellcommunication. Administration of anti-connexin polynucleotide(s) willmodulate communication between the cells and minimize additional cellloss or injury or consequences of injury.

While the delivery period will be dependent upon both the site at whichthe downregulation is to be induced and the therapeutic effect which isdesired, continuous or slow-release delivery for about 0.5 hours, about1-2 hours, about 2-4 hours, about 4-6 hours, about 6-8, or about 24hours or longer is provided. In accordance with the present invention,this maybe achieved by inclusion of the anti-connexin polynucleotides(with or without one or more therapeutic agents or agents useful forwound healing) in a formulation together with a pharmaceuticallyacceptable carrier or vehicle, particularly in the form of a formulationfor continuous or slow-release administration.

The routes of administration and dosages described herein are intendedonly as a guide since a skilled physician will determine the optimumroute of administration and dosage for any particular patient.

Any of the methods of treating a subject having or suspected of havingor a disease, disorder, or condition referenced or described herein mayutilize the administration of any of the doses, dosage forms,formulations, and/or compositions herein described.

Dressings and Matrices

In one aspect, one or more anti-connexin polynucleotides either alone orin combination with one or more therapeutic agents useful in woundhealing are provided in the form of a dressing or matrix. In certainembodiments, the one or more agents of the invention are provided in theform of a liquid, semi solid or solid composition for applicationdirectly, or the composition is applied to the surface of, orincorporated into, a solid contacting layer such as a dressing gauze ormatrix. The dressing composition may be provided for example, in theform of a fluid or a gel. One or more anti-connexin polynucleotides andone or more anti-connexin peptides or peptidomimetics may be provided incombination with conventional pharmaceutical excipients for topicalapplication. Suitable carriers include: Pluronic gels, Polaxamer gels,Hydrogels containing cellulose derivatives, including hydroxyethylcellulose, hydroxymethyl cellulose, carboxymethyl cellulose,hydroxypropylmethyl cellulose and mixtures thereof; and hydrogelscontaining polyacrylic acid (Carbopols). Suitable carriers also includecreams/ointments used for topical pharmaceutical preparations, e.g.,creams based on cetomacrogol emulsifying ointment. The above carriersmay include alginate (as a thickener or stimulant), preservatives suchas benzyl alcohol, buffers to control pH such as disodium hydrogenphosphate/sodium dihydrogen phosphate, agents to adjust osmolarity suchas sodium chloride, and stabilizers such as EDTA.

In addition to the biological matrices previously mentioned, suitabledressings or matrices may include, for example, the following with oneor more anti-connexin polynucleotides and one or more other therapeuticagents.

1) Absorptives: suitable absorptives may include, for example,absorptive dressings, which can provide, for example, a semi-adherentquality or a non-adherent layer, combined with highly absorptive layersof fibers, such as for example, cellulose, cotton or rayon.Alternatively, absorptives may be used as a primary or secondarydressing.

2) Alginates: suitable alginates include, for example, dressings thatare non-woven, non-adhesive pads and ribbons composed of naturalpolysaccharide fibers or xerogel derived from seaweed. Suitablealginates dressings may, for example, form a moist gel through a processof ion exchange upon contact with exudate. In certain embodiments,alginate dressings are designed to be soft and conformable, easy topack, tuck or apply over irregular-shaped areas. In certain embodiments,alginate dressings may be used with a second dressing.

3) Antimicrobial Dressings: suitable antimicrobial dressings mayinclude, for example, dressings that can facilitate delivery ofbioactive agents, such as, for example, silver and polyhexamethylenebiguanide (PHMB), to maintain efficacy against infection, where this isneeded or desirable. In certain embodiments, suitable antimicrobialdressings may be available as for example, as sponges, impregnated wovengauzes, film dressings, absorptive products, island dressings, nylonfabric, non-adherent barriers, or a combination of materials.

4) Biological & Biosynthetics: suitable biological dressings orbiosynthetic dressings may include, for example, gels, solutions orsemi-permeable sheets derived from a natural source, e.g., pigs or cows.In certain embodiments, a gel or solution is applied to the treatmentsite and covered with a dressing for barrier protection. In anotherembodiment, a biological-based (e.g., pig intestinal mucosa or bladdertissue) or biosynthetic-based sheet is placed in situ which may act asmembrane, remaining in place after a single application, or the may bebiological dressings or biosynthetic dressings may be prepared inadvance to include one or more, preferably two, anti-connexin agents.

5) Collagens: suitable collagen dressings may include, for example,gels, pads, particles, pastes, powders, sheets or solutions derived fromfor example, bovine, porcine or avian sources or other natural sourcesor donors. In certain embodiments, the collagen dressing may interactwith treatment site exudate to form a gel. In certain embodiments,collagen dressing may be used in combination with a secondary dressing.

6) Composites: suitable composite dressings may include, for example,dressings that combine physically distinct components into a singleproduct to provide multiple functions, such as, for example, a bacterialbarrier, absorption and adhesion. In certain embodiment, the compositedressings are comprised of, for example, multiple layers and incorporatea semi- or non-adherent pad. In certain embodiment, the composite mayalso include for example, an adhesive border of non-woven fabric tape ortransparent film. In certain other embodiment, the composite dressingmay function as for example, either a primary or a secondary dressingand in yet another embodiment, the dressing may be used in combinationwith topical pharmaceutical composition.

7) Contact Layers: suitable contact layer dressings may include, forexample, thin, non-adherent sheets placed on an area to protect tissuefrom for example, direct contact with other agents or dressings appliedto the treatment site. In certain embodiments, contact layers may bedeployed to conform to the shape of the area of the treatment site andare porous to allow exudate to pass through for absorption by anoverlying, secondary dressing. In yet another embodiment, the contactlayer dressing may be used in combination with topical pharmaceuticalcomposition.

8) Elastic Bandages: suitable elastic bandages may include, for example,dressings that stretch and conform to the body contours. In certainembodiment, the fabric composition may include for example, cotton,polyester, rayon or nylon. In certain other embodiments, the elasticbandage may for example, provide absorption as a second layer ordressing, to hold a cover in place, to apply pressure or to cushion atreatment site.

9) Foams: suitable foam dressings may include, for example, sheets andother shapes of foamed polymer solutions (including polyurethane) withsmall, open cells capable of holding fluids. Exemplary foams may be forexample, impregnated or layered in combination with other materials. Incertain embodiment, the absorption capability may be adjusted based onthe thickness and composition of the foam. In certain other embodiments,the area in contact with the treatment site may be non-adhesive for easyremoval. In yet another embodiment, the foam may be used in combinationwith an adhesive border and/or a transparent film coating that can serveas an anti-infective barrier.

10) Gauzes & Non-Woven dressings: suitable gauze dressings and wovendressings may include, for example, dry woven or non-woven sponges andwraps with varying degrees of absorbency. Exemplary fabric compositionmay include, for example, cotton, polyester or rayon. In certainembodiment, gauzes and non-woven dressing may be available sterile ornon-sterile in bulk and with or without an adhesive border. Exemplarygauze dressings and woven dressings may be used for cleansing, packingand covering a variety of treatment sites.

11) Hydrocolloids: suitable hydrocolloid dressings may include, forexample, wafers, powders or pastes composed of gelatin, pectin orcarboxymethylcellulose. In certain embodiment, wafers are self-adheringand available with or without an adhesive border and in a wide varietyof shapes and sizes. Exemplary hydrocolloids are useful on areas thatrequire contouring. In certain embodiments, powders and pasteshydrocolloids may use used in combination with a secondary dressing.

12) Hydrogels (Amorphous): suitable amorphous hydrogel dressings mayinclude, for example, formulations of water, polymers and otheringredients with no shape, designed to donate moisture and to maintain amoist healing environments and or to rehydrate the treatment site. Incertain embodiment, hydrogels may be used in combination with asecondary dressing cover.

13) Hydrogels: Impregnated Dressings: suitable impregnated hydrogeldressings may include, for example, gauzes and non-woven sponges, ropesand strips saturated with an amorphous hydrogel. Amorphous hydrogels mayinclude for example, formulations of water, polymers and otheringredients with no shape, designed to donate moisture to a drytreatment site and to maintain a moist healing environment.

14) Hydrogel Sheets: suitable hydrogel sheets may include for example,three-dimensional networks of cross-linked hydrophilic polymers that areinsoluble in water and interact with aqueous solutions by swelling.Exemplary hydrogels are highly conformable and permeable and can absorbvarying amounts of drainage, depending on their composition. In certainembodiment, the hydrogel is non-adhesive against the treatment site ortreated for easy removal.

15) Impregnated Dressings: suitable impregnated dressings may include,for example, gauzes and non-woven sponges, ropes and strips saturatedwith a solution, an emulsion, oil, gel or some other pharmaceuticallyactive compound or carrier agent, including for example, saline, oil,zinc salts, petrolatum, xeroform and scarlet red as well as thecompounds described herein.

16) Silicone Gel Sheets: suitable silicone gel sheet dressings mayinclude, for example, soft covers composed of cross-linked polymersreinforced with or bonded to mesh or fabric.

17) Solutions: suitable liquid dressings may include, for example,mixtures of multiprotein material and other elements found in theextracellular matrix. In certain embodiment, exemplary solutions may beapplied to the treatment site after debridement and cleansing and thencovered with an absorbent dressing or a nonadherent pad.

18) Transparent Films: suitable transparent film dressings may includepolymer membranes of varying thickness coated on one side with anadhesive. In certain embodiments, transparent films are impermeable toliquid, water and bacteria but permeable to moisture vapor andatmospheric gases. In certain embodiments, the transparency allowsvisualization of the treatment site.

19) Fillers: suitable filler dressings may include, for example, beads,creams, foams, gels, ointments, pads, pastes, pillows, powders, strandsor other formulations. In certain embodiment, fillers are non-adherentand may include a time-released antimicrobial. Exemplary fillers may beuseful to maintain a moist environment, manage exudate, and fortreatment of for example, partial- and full-thickness wounds, infectedwounds, draining wounds and deep wounds that require packing.

Treatment

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for fibrosis, and various fibrotic diseases,disorders, or conditions characterized in whole or in part by (1)fibrous material, (2) excess production of fibrous material within theextracellular matrix, and/or (3) replacement of normal tissue elementsby abnormal, non-functional, and/or excessive accumulation ofmatrix-associated components, comprising administering a compositioncomprising an anti-connexin polynucleotide and a pharmaceuticallyacceptable carrier or diluent. Preferred anti-connexin polynucleotidesare anti-connexin 43 polynucleotides.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for fibrosis, comprising administering acomposition comprising an anti-connexin polynucleotide, e.g., ananti-connexin 43 polynucleotide, and a pharmaceutically acceptablecarrier or diluent.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for fibrosis, comprising administering acomposition comprising an anti-connexin polynucleotide, e.g., ananti-connexin 43 polynucleotide, and a pharmaceutically acceptablecarrier or diluent.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for pulmonary fibrosis, comprisingadministering a composition comprising an anti-connexin polynucleotide,e.g., an anti-connexin 43 polynucleotide, and a pharmaceuticallyacceptable carrier or diluent. In one embodiment, pulmonary fibrosis isdiffuse interstitial pulmonary fibrosis. In another embodiment, thepulmonary fibrosis is glomerulosclerosis pulmonary fibrosis, idiopathicpulmonary fibrosis, silicosis, asbestosis, and chemotherapy/radiationinduced pulmonary fibrosis.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for kidney fibrosis, comprising administeringa composition comprising an anti-connexin polynucleotide, e.g., ananti-connexin 43 polynucleotide, and a pharmaceutically acceptablecarrier or diluent. In one embodiment the kidney fibrosis is associatedwith glomerular sclerosis, renal tubulointerstitial fibrosis, orprogressive renal disease. In one embodiment kidney fibrosis isassociated with diabetic neuropathy.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for liver fibrosis, comprising administeringa composition comprising an anti-connexin polynucleotide, e.g., ananti-connexin 43 polynucleotide, and a pharmaceutically acceptablecarrier or diluent. In one embodiment the liver fibrosis arises fromchronic liver injury. In one embodiment, liver fibrosis is associatedwith haemochromatosis, Wilson's disease, alcoholism, schistosomiasis,viral hepatitis, bile duct obstruction, exposure to toxins, andmetabolic disorders.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for liver cirrhosis, comprising administeringa composition comprising an anti-connexin polynucleotide, e.g., ananti-connexin 43 polynucleotide, and a pharmaceutically acceptablecarrier or diluent.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for cardiac fibrosis, comprisingadministering a composition comprising an anti-connexin polynucleotide,e.g., an anti-connexin 43 polynucleotide, and a pharmaceuticallyacceptable carrier or diluent. In one embodiment, the cardiac fibrosisis endocardial fibrosis. In another embodiment, the cardiac fibrosis isendomyocardial fibrosis.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for oral submucous fibrosis, comprisingadministering a composition comprising an anti-connexin polynucleotide,e.g., an anti-connexin 43 polynucleotide, and a pharmaceuticallyacceptable carrier or diluent.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for retroperitoneal fibrosis, comprisingadministering a composition comprising an anti-connexin polynucleotide,e.g., an anti-connexin 43 polynucleotide, and a pharmaceuticallyacceptable carrier or diluent.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for deltoid fibrosis, comprisingadministering a composition comprising an anti-connexin polynucleotide,e.g., an anti-connexin 43 polynucleotide, and a pharmaceuticallyacceptable carrier or diluent.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, methods of treating a subject having or suspected of having orpredisposed to, or at risk for acute fibrosis, comprising administeringa composition comprising an anti-connexin polynucleotide, e.g., ananti-connexin 43 polynucleotide, and a pharmaceutically acceptablecarrier or diluent. In certain embodiments, the acute fibrosis isassociated with various forms of trauma including, for example,accidental injuries, infections, radiation or chemotherapy treatments.

The invention includes methods for treating and/or preventing, in wholeor in part, various diseases, disorders and conditions, including, forexample, contracture or recurrence thereof, comprising administering aeffective amount of a composition comprising an anti-connexinpolynucleotide. In one embodiment the contracture is capsularcontracture, Dupytren's contracture, Volkmann's contracture,Ledderhose's contracture, Peyronie's contracture. In one embodiment, thecomposition is administered to the site of the injury before, at thetime of and/or after a release procedure (e.g., forced manipulation,open release, arthroscopic release, or debulking of scar) to prevent therecurrence of scarred and abnormal tissue and/or further contracture.

In one embodiment any one of the methods of treatment described hereinfurther comprises administration of second composition having one ormore drugs effective in preventing and/or decreasing fibrosis orfibrotic tissue. In one aspect the second composition comprises one ormore anti-connexin polynucleotides, e.g., one or more anti-connexin 43polynucleotides. In one aspect the second composition comprises one ormore therapeutic agents. In another aspect of the invention, the secondcomposition comprises one or more agents useful for wound healing. Inanother aspect of the invention, the second composition comprises one ormore one or more therapeutic agents.

When not administered as a fixed combination, preferred methods includethe sequential administration of one or more anti-connexinpolynucleotides and one or more therapeutic agents and/or agents usefulfor wound healing. Preferably, the polynucleotides and agents areadministered sequentially within at least about one-half hour of eachother. The polynucleotides and agents may also be administered withabout one hour of each other, with about one to two or three days toabout one week of each other, or as otherwise deemed appropriate.Preferably, the anti-connexin polynucleotide is administered first.

In another embodiment for treatment of fibrosis and fibrotic diseases,disorders and conditions, either or both of the one or moreanti-connexin polynucleotides and one or more therapeutic agents and/oragents useful for wound healing are provided in amounts or doses thatare less that those used when the polynucleotides or agents areadministered alone, i.e., when they are not administered in combination.Such lesser amounts of agents administered are typically from aboutone-twentieth to about one-tenth the amount or amounts of the agent whenadministered alone, and may be about one-eighth the amount, aboutone-sixth the amount, about one-fifth the amount, about one-fourth theamount, about one-third the amount, and about one-half the amount whenadministered alone.

Compositions

The present invention is directed to pharmaceutical compositions andformulations useful in treating or preventing fibrosis and fibroticdiseases, disorders and conditions, wherein the composition orformulation comprises therapeutically effective amounts of ananti-connexin polynucleotide, such as a connexin antisensepolynucleotide.

Equally, in instances of other tissue damage the methods, compositionsand formulations of the invention are effective in treating orpreventing fibrosis. The compositions and formulations, therefore, haveclear benefit in the treatment of fibrosis and fibrotic conditions.

In one preferred form, the composition contains one or moreanti-connexin polynucleotides, for example a connexin antisensepolynucleotide, to the mRNA of one connexin protein only. Mostpreferably, this connexin protein is connexin 43.

Alternatively, the compositions may comprise polynucleotides to morethan one connexin protein. Preferably, one of the connexin proteins towhich polynucleotides are directed is connexin 43. Other connexinproteins to which oligodeoxynucleotides are directed may include, forexample, connexins 26, 30, 31.1, 32, and 37. Suitable exemplarypolynucleotides (and ODNs) directed to various connexins are set forthin Table 1.

Many aspects of the invention are described with reference tooligodeoxynucleotides. However it is understood that other suitablepolynucleotides (such as RNA polynucleotides) may be used in theseaspects. Other anti-connexin oligonucleotides are RNAi and siRNAoligonucleotides.

Accordingly, in one aspect, the invention provides compositions for usein therapeutic treatment, which comprises at least one anti-connexinpolynucleotide, preferably an anti-connexin 43 polynucleotide. In apreferred embodiment, the composition further comprises apharmaceutically acceptable carrier or vehicle.

Kits, Medicaments and Articles of Manufacturer

In one aspect, the invention provides a kit for preventing or treatingfibrosis.

The kit may include one or more compositions described herein. Forexample, the kit may include a composition comprising an effectiveamount of one or more anti-connexin polynucleotides, e.g., ananti-connexin 43 polynucleotides, effective for the treatment of asubject having, at risk for, or predisposition to a fibrotic disease,disorder or condition. In one embodiment, the kit comprises acomposition that comprises an effective amount of one or morepolynucleotide homologues effective for the treatment of a subjecthaving, at risk for, or predisposition to a fibrotic disease, disorderor condition.

Optionally, one or more anti-connexin polynucleotides may also be usedin the manufacture of the medicament useful for the treatment of of asubject having, at risk for, or predisposition to a fibrotic disease,disorder or condition. In one embodiment, the medicament comprises atherapeutically effective amount of an anti-connexin polynucleotide,preferably an anti-connexin 43 polynucleotide, and a pharmaceuticallyacceptable carrier.

In another aspect, the invention includes an article of manufacturecomprising a vessel containing an effective amount of one or moreanti-connexin polynucleotides, e.g., an anti-connexin 43 polynucleotide,and instructions for use, including use for the treatment of a subjecthaving, at risk for, or predisposition to a fibrotic disease, disorderor condition.

Thus, in accordance with the invention, there are provided compounds,compositions, formulations by which cell-cell communication can beregulated or downregulated in a transient and site-specific manner. Theformulations therefore have application in methods of therapy and inother treatments.

In instances of tissue damage or fibrosis, the compounds, compositionsand formulations of the invention will be effective in both preventingfibrosis and promoting the healing process where needed. Theformulations therefore will have benefit in the prevention of fibrosis,whether the result of external trauma or disease state, for example.

A better understanding of the invention will be gained by reference tothe following experimental section. The following experiments areillustrative and are not intended to limit the invention or the claimsin any way.

EXAMPLES Example 1

This example demonstrates a method for qualitative and quantitativeevaluation of anti connexin agents and their ability to inhibitfibrosis. Rats are injected either with anti-thymocyte serum (ATS) (seeS. Okuda et al., J. Clin. Invest., Vol. 86, (1990, pp. 453-462) toinduce glomerulonephritis or with phosphate buffered saline (PBS) toserve as controls. Six days later, the kidneys are removed, and theglomeruli are isolated and placed in culture for 72 hours. Cultureconditions consist of 2000 glomeruli/well in a 2 ml volume of serum-freeRPMI 1640 (with insulin supplementation) (Gibco; Gaithersburg, Md.).Test anti-connexin polynucleotides are added at the time of the culture.The supernatant from the cultures is collected and stored at −70° C.until assayed to determine the concentration of collagen I, transforminggrowth factor β-1 (TGFβ-1), fibronectin containing an extra domain A(fibronectin EDA+), and plasminogen activator inhibitor I 9PAI-I) asmarkers of fibrotic activity. In addition, individual glomeruli areexamined by immunofluorescent staining and scored for relevant matrixproteins. Values are compared between PBS-treated, negative fibroticcontrol glomeruli; ATS-treated, non-drug treated, positive fibroticcontrol glomeruli; and the ATS-treated, drug treated, fibrotic glomerulito determine the degree to which the fibrotic process is inhibited bythe anti-connexin polynucleotide.

Example 2

This example demonstrates a method for testing anti connexin agents andtheir ability to inhibit fibrosis. Rats are injected either withanti-thymocyte serum (ATS) to induce glomerulonephritis or withphosphate buffered saline (PBS) as a control. One hour later, treatmentis initiated with anti-connexin polynucleotide. Anti-connexinpolynucleotides are administered subcutaneously twice per day for 5days. On day 5 the rats are placed in metabolic cages, and 24 hour urineis collected to determine protein content. On day 6, the kidneys areremoved, and tissue samples are either placed in formalin or frozen forhistological evaluation. Glomeruli are isolated from the remainingtissue and are placed in culture for 72 hours. Culture conditionsconsist of 2000 glomeruli/well in a 1 ml volume of serum free RPMI 1640(with insulin supplementation). The supernatant from the cultures arecollected and stored at −70° C., until assayed to determine theconcentration of collagen I, transforming growth factor β-1 (TGF β-1),fibronectin containing an extra domain A (fibronectin EDA+), andplasminogen activator inhibitor 1 (PAI1) as markers of fibroticactivity. The presence of matrix proteins is measured viaimmunofluorescent staining of frozen kidney sections with antibodies tomatrix proteins induced by TGF β-1 such as fibronectin EDA+, collagen I,PAI1, and tenasin. From the cultured isolated glomeruli directmeasurements of TGF β-1, PAI1, and fibronectin secreted into the culturesupernatant can be determined via ELISAs (enzyme-linked immunoabsorbentassay). Glomeruli from samples in each group can be used to extract mRNAand the message levels for TGF β-1, GADPH, collagen I, collagen III,Fibronectin, and PAI1 determined by Northern analysis. As an indicatorof gross histological changes, PAS (periodic acid-Schiff) stainedparaffin sections are graded on the basis of their pathological matrixscores. Values are compared between PBS-treated, negative fibroticcontrol animals; ATS-treated, non-drug treated, drug-treated animals todetermine the degree to which the fibrotic process is inhibited by theanti-connexin polynucleotide.

Example 3

Wounds are created in C57BL6/KsJ db/db mice with a 4 mm biopsy punch.The mice may be obtained from The Jackson Laboratories and are typicallyaged 3-7 months before the onset of the wounding protocol. All mice areanesthetized prior to wounding. Two wounds are introduced onto the upperback of each animal by pulling the skin away from underlying structuresand pushing the punch through the isolated skin. Typically, wounds arecreated to an average depth of 1.7 mm, with a range of 1.3 to 2.2 mm. Nomuscle involvement occurred during the course of wounding. Immediatelypost-wounding the wounds are either treated with normal saline (to serveas the non treated control group) or with suitable test anti-connexinpolynucleotide.

Each day the wounds are digitally photographed and wound areas aredetermined by computer integration of the photographs. All woundtreatments and the subsequent data analysis are performed in a blindmanner. Wound area at the time of wounding (day 0) is set to a relativevalue of 1 for all wounds; such that subsequent wound areas areconverted to relative wound areas by dividing the wound area at day “n”by the wound area at day zero.

TAnti-fibrotic efficacy based on the application of a single dose of theanti-connexin polynucleotide (at the time of wounding, day zero) asdetermined by the time to full wound closure in a mouse model isdetermined.

Endpoints such as fibrosis, wound closure, wound contraction, andinflammation are assessed starting at day one post wounding andcontinues for a pre-determined period of time (e.g. hours, days, weeks,months or years).

Example 4

The anti-fibrotic efficacy of anti-connexin polynucleotide is assessedin a mouse model of fibrosis treatment. Suitable numbers of adult miceare divided into statistically meaningful sample size groups (e.g. sixgroups in groups of eight): four treated and four control. The mice areanaesthetized using IP injection of Avertine, the dorsal surface shavedand two 1 cm incisions made through the skin down to and including thepanniculus carnosus muscle at specific anatomical positions. The woundsare left unsutured and the animals returned to individual cages. Groupof animals are killed after 1 day (d), 3 d, 5 d, 7 d, 14 and 70 dpost-wounding and the wounds harvested. Half of each harvested wound isfixed in formal saline and the other half embedded in OCT medium andfrozen over liquid nitrogen. Photographic records are kept of the woundsat each time point to enable comparison of microscopic and macroscopicresults.

Histological Assessment:

Haematoxylin and Eosin (H&E) and Masson's Trichrome stains are used todetermine the cellularity and collagen content of the woundsrespectively.

Scoring of Fibrosis:

At 70 Days Post Wounding, the histology slides are scored using a VisualAnalogue Scale (VAS) consisting of a 10 cm line where 0 representsnormal skin and 10 an extreme case of fibrosis. A ranking scale is alsoused wherein 0 represents normal skin and 5 an extreme fibrotic skin.The ranking of 3 is used for the score of a control fibrosis.

Immunohistochemistry:

Samples from 1, 3, 5 and 7 day wounds are stained using severalantibodies including: 1) Anti-mouse fibronectin or 2) TRITC-labelledphalloidin. Phalloidin is extracted from the mushroom amanita phalloidesand binds to filamentous actin (F-actin) so is useful in localizing anddistinguishing between extra- and intracellular F-actin.

Image Analysis:

Image analysis is carried out using PC based image capture system (‘PCImage’) and the following parameters are measured in order to quantifydifferences in the anti-fibrotic efficacy of the test agent between testand control wounds: 1) Wound width (both linear between wound edges andactual perimeter); 2) Retraction of the panniculus carnosus muscle; 3)Mid-wound width; 4) Re-epithelialization; 5) Fibrosis width at 3 points:base, middle and top; and 6) Thickness of new epithelium.

All wounds are measured for wound width and retraction of the panniculuscarnosus muscle, the other measurements are taken at appropriate timepoints. Statistical analysis of the measurements is performed usingsuitable statistical software well-known and widely available in theart. Exemplary statistical tests may include Mann-Whitney U test and theKolgomorov-Smirnov test to compare results from the control and testanimals.

Histology:

Histological assessment and endpoints for fibrosis may include, forexample, evidence of neovasculorisation and collagen formation in thewound area; inflammation, level of new collagen formation; localaccumulation of hair follicles immediately adjacent to and surroundingthe fibrosis; and improvements in the quality of fibrotic tissue.

Example 5

This example describes and internal wound healing dressing/filmcomprising encapsulation of anti-connexin polynucleotides in EthyleneVinyl Acetate Films and Polycaprolactone Paste.

Suitable amounts of an exemplary anti-connexin polynucleotide and 45 mgof ethylene vinyl acetate (EVA, molecular weight approximately 50 k,Polysciences) are dissolved/suspended in 1 ml of dicloromethane. Twohundred μl of the solution is pipetted onto 1 cm diameter teflon discsand allowed to dry overnight (solvent evaporation) to form thin elasticfilms to give approximately 10 mg films with an approximate thickness of100 μm.

The rate of drug release from these films is measured by placing 5 mgsections of films in 20 ml capped glass tubes containing 10 ml ofphosphate buffered saline (PBS) pH 7.4. The tubes are capped, and placedin an orbital shaker at 37° C. At specified times, the tubes are removedand the amount of drug released is analysed by absorbance spectroscopy.This and/or other exemplary dosage form of anti-connexin polynucleotiderepresents a biocompatible, biodegradable, injectable formulation of thedrug that releases the drug in a controlled manner.

PCL paste: exemplary anti-connexin polynucleotide is blended intopolycaprolactone (PCL, Birmingham polymers, molecular weight 54K) at 60°C. by spatula levigation at a concentration of 10% w/w. This mixture isthen pipetted into 1 ml plastic syringes and allowed to cool. Thisformulation could be injected through an 18 gauge needle at 56° C.

To measure drug release from the PCL paste, 10 mg aliquots of moltenpaste are injected onto the base of 15 ml glass tubes and allowed tocool and set. Fifteen ml of PBS is added to each tube and the tubes arecapped, and tumbled end over end in a 37° C. oven. At specified times,the tubes are removed and the amount of drug released is analysed byabsorbance spectroscopy. The release profile of the anti-connexinpolynucleotide is obtained. This and/or other exemplary dosage form ofanti-connexin polynucleotide represents a biocompatible, biodegradable,injectable formulation of the drug that releases the drug in acontrolled manner.

Example 6

This example describes membranes loaded with anti-connexinpolynucleotides.

Medical grade sodium hyaluronate is obtained from Lifecore Scientific.All solvents are HPLC grade and obtained from Fisher. Plastic Petridishes are obtained from Fisher Scientific. Ethyl-3-(dimethylamino)carbodiimide (EDAC) and anti-connexin polynucleotide are prepared asdescribed elsewhere throughout the instant disclosure.

Preparation of Films. Anti-connexin polynucleotide loaded films are madeby preparing an exemplary solution of 0.6% w/v anti-connexinpolynucleotide, 0.4% w/v sodium hyaluronate and 0.15% w/v glycerol inwater. Control films (no anti-connexin polynucleotide) are made bypreparing a solution or mixture of 0.4% w/v sodium hyaluronate and 0.15%w/v glycerol in water. Anti-connexin polynucleotide loaded films andcontrol films are cast from these solutions by pipetting 4 g of eachsolution into separate 2.5 cm diameter plastic Petri dishes and dryingfor 24 hours at 60° C. The crosslinking agent EDAC is included at 4 mM(final concentration). Each dried film is then carefully removed fromthe Petri dish using a surgical blade.

Sterilization. Films are packed between 5 cm×5 cm weighing paper (Fisherscientific) and heat sealed in plastic bags. Films are then terminallysterilized using gamma irradiation from a cobalt-60 source and exposedto 2.5 Mrad of radiation with cooling of the sealed tube on ice.

Example 7

This example describes an external wound dressing comprising ananti-connexin polynucleotide. Drug loading into fatty acid (e.g. fishoil) derived membranes for wound dressing. Pure fish oil is heated at200° F. to obtain a viscosity of 15,000-20,000 cps at 24° C. to form apre-treated or pre-thickened fish oil. 3.1 g of the pre-treated orpre-thickened fish oil is then mixed with an appropriate amount of anexemplary anti-connexin polynucleotide. The mixture is then heatedgently to allow the anti-connexin polynucleotide to dissolve in the fishoil. This resulted in an anti-connexin polynucleotide in fish oilformulation by weight. After heating, the mixture is cast onto a Teflonmat with a casting knife to form a thin film. The thin film is thenplaced under a UV lamp for 15 minutes. After exposure to UV light, thethin film is heated in an oven to heat cure, after which the thin filmis removed from the oven and allowed to cool for 1 hour. After the thinfilm is cooled, it is peeled from the Teflon mat to form a stand-alonefilm. The resultant film had a thickness of approximately 0.005″. Drugextraction and dissolution are performed on the film by high performanceliquid chromatography (HPLC). The extraction result can indicate amountof drug loading per film sample length. In general, the dissolution ofthe anti-connexin polynucleotide should release the drug in anapproximately linear fashion as a function of time.

Example 8

This example describes overlaying a drug-loaded fish oil on astand-alone film for use in treating or preventing fibrosis. Pure fishoil is heated to obtain a viscosity greater than 100,000 cps at 24° C.to form the pre-cured fish oil film. 3.33 g of pre-cured fish oil ismixed with an appropriate amount of anti-connexin polynucleotide to forma mixture. This resulted in a fish oil formulation. After theanti-connexin polynucleotide is solubilized in the pre-cured fish oil,the mixture is brushed onto a 1″ by 1½″ piece of stand-alone film. Thefilm with the drug coating is then heated. Drug extraction anddissolution are performed on the film by HPLC. The extraction result canindicate amount of drug loading per film sample length. In general, thedissolution of the anti-connexin polynucleotide should release the drugin an approximately linear fashion as a function of time.

Example 9

This example describes drug coating by allowing a stand-alone woundcovering film to swell with a solution including a therapeuticanticonnexin agent. Suitable amounts of anti-connexin polynucleotidesare mixed with suitable amounts of EtOH. This resulted in a %formulation by weight. A 1″ by 1½″ of stand-alone film is dipped intothe anti-connexin polynucleotide formulation and allowed to swell. Thestand-alone film is then allowed to air dry. The resultant film isapproximately 0.005″ in thickness. Drug extraction and dissolution areperformed on the film by HPLC. The extraction result can indicate amountof drug loading per film sample length. In general, the dissolution ofthe anti-connexin polynucleotide should release the drug in anapproximately linear fashion as a function of time.

Example 10

Anti-connexin agent is conveniently formulated in a form suitable foradministration according to the methods of the present invention.

Suitable formulations include a mixture of the following formulatingagents. The amount of the individual anti-connexin agent or agents andformulating agents will depend on the particular use intended.

ASO in PBS Polyquarternium 10 HEC/HPMC/CMC Na Hyaluronate Tween 20Poloxamer 188 Pluronic 87 NF SLES Poly L-lysine/Polyethylene ImineBanzalkonium chloride Methyl paraben Propl paraben Propylene Glycol 10mM Phosphate Buffer

Example 11

Formulations for use according to methods of the present invention areprepared by mixing the compounds in the proportions noted below. In onepreferred embodiment, the anti-connexin agent is an anti-connexinpolynucleotide. In other embodiments, the anti-connexin polynucleotideis an anti-sense oligonucleotide, for example, an anti-senseoligonucleotide of SEQ. ID. NO. 1

Formulation A

Made up of the following materials (% w/w)—Anti-connexin agent inphosphate-buffered saline (0.47%); Methylparaben (0.17%); Propylparaben(0.03%); Propylene Glycol (1.5%); HPMC (1.5%); and 10 mM PhosphateBuffer (96.33%). Formulation is a clear gel with pH ˜6.74 and osmolalityof 244.

Formulation B

Made up of the following materials (% w/w)—Anti-connexin agent inphosphate-buffered saline (0.47%); Methylparaben (0.17%); Propylparaben(0.03%); Propylene Glycol (1.5%); HPMC (1.5%); 0.5% BAC (0.1%); and 10mM Phosphate Buffer (96.23%). Formulation is a clear gel with pH ˜6.65and osmolality of 230.

Formulation C

Made up of the following materials (% w/w)—Anti-connexin agent inphosphate-buffered saline (0.47%); Methylparaben (0.17%); Propylparaben(0.03%); Propylene Glycol (1.5%); HPMC (1.5%); Polyquaternium 10 (0.5%);Poloxamer 188 (0.1%); and 10 mM Phosphate Buffer (95.73%). Formulationis a slightly hazy gel with pH ˜6.59 and osmolality of 233.

Formulation D

Made up of the following materials (% w/w)—Anti-connexin agent inphosphate-buffered saline (0.47%); Methylparaben (0.17%); Propylparaben(0.03%); Propylene Glycol (1.5%); HPMC (1.5%); SLES (0.5%); and 10 mMPhosphate Buffer (95.83%). Formulation is a clear gel with pH ˜6.8 andosmolality of 246.

Formulation E

Made up of the following materials (% w/w)—Anti-connexin agent inphosphate-buffered saline (0.47%); Methylparaben (0.17%); Propylparaben(0.03%); Propylene Glycol (1.5%); HPMC (1.5%); Poloxamer 188 (0.1%); 25KPolyethylene Imine (0.075%); and 10 mM Phosphate Buffer (96.155%).Formulation is a hazy gel with pH ˜7.8 and osmolality of 249.

Formulation F

Made up of the following materials (% w/w)—Anti-connexin agent inphosphate-buffered saline (0.47%); Methylparaben (0.17%); Propylparaben(0.03%); Propylene Glycol (1.5%); HPMC (1.5%); Sodium Hyaluronate(0.1%); and 10 mM Phosphate Buffer (96.23%). Formulation is a clear gelwith pH ˜6.88 and osmolality of 289.

Formulation G

Made up of the following materials (% w/w)—Anti-connexin agent inphosphate-buffered saline (0.47%); Methylparaben (0.17%); Propylparaben(0.03%); Propylene Glycol (1.5%); Sodium Hyaluronate (1.0%); and 10 mMPhosphate Buffer (96.83%). Formulation is a clear gel with pH ˜6.81 andosmolality of 248.

All patents, publications, scientific articles, web sites, and otherdocuments and materials referenced or mentioned herein are indicative ofthe levels of skill of those skilled in the art to which the inventionpertains, and each such referenced document and material is herebyincorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such patents, publications, scientific articles,web sites, electronically available information, and other referencedmaterials or documents.

The written description portion of this patent includes all claims.Furthermore, all claims, including all original claims as well as allclaims from any and all priority documents, are hereby incorporated byreference in their entirety into the written description portion of thespecification, and Applicants reserve the right to physicallyincorporate into the written description or any other portion of theapplication, any and all such claims. Thus, for example, under nocircumstances may the patent be interpreted as allegedly not providing awritten description for a claim on the assertion that the precisewording of the claim is not set forth in haec verba in writtendescription portion of the patent.

The claims will be interpreted according to law. However, andnotwithstanding the alleged or perceived ease or difficulty ofinterpreting any claim or portion thereof, under no circumstances mayany adjustment or amendment of a claim or any portion thereof duringprosecution of the application or applications leading to this patent beinterpreted as having forfeited any right to any and all equivalentsthereof that do not form a part of the prior art.

All of the features disclosed in this specification may be combined inany combination. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Thus,from the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention. Other aspects, advantages,and modifications are within the scope of the following claims and thepresent invention is not limited except as by the appended claims.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,the terms “comprising”, “including”, “containing”, etc. are to be readexpansively and without limitation. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by various embodiments and/or preferredembodiments and optional features, any and all modifications andvariations of the concepts herein disclosed that may be resorted to bythose skilled in the art are considered to be within the scope of thisinvention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

It is also to be understood that as used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise, the term “X and/or Y”means “X” or “Y” or both “X” and “Y”, and the letter “s” following anoun designates both the plural and singular forms of that noun. Inaddition, where features or aspects of the invention are described interms of Markush groups, it is intended, and those skilled in the artwill recognize, that the invention embraces and is also therebydescribed in terms of any individual member and any subgroup of membersof the Markush group, and applicants reserve the right to revise theapplication or claims to refer specifically to any individual member orany subgroup of members of the Markush group.

Other embodiments are within the following claims. The patent may not beinterpreted to be limited to the specific examples or embodiments ormethods specifically and/or expressly disclosed herein. Under nocircumstances may the patent be interpreted to be limited by anystatement made by any Examiner or any other official or employee of thePatent and Trademark Office unless such statement is specifically andwithout qualification or reservation expressly adopted in a responsivewriting by Applicants.

1-35. (canceled) 36.-54. (canceled)
 55. A method of preventing ordecreasing fibrosis of the eye in a subject in need thereof, the methodcomprising administering to the subject an anti-connexin polynucleotide.56. The method of claim 55, wherein said anti-connexin polynucleotide isan antisense polynucleotide selected from the group consisting ofantisense oligonucleotides, deoxyribozymes, morpholino oligonucleotides,RNAi molecules, siRNA molecules, PNA molecules, DNAzymes, and5′-end-mutated U1 small nuclear RNAs, and analogs of the preceding. 57.The method of claim 55, wherein the anti-connexin polynucleotide isadministered to prevent or retard fibrosis.
 58. The method of claim 55,wherein the subject has fibrotic macular degeneration.
 59. The method ofclaim 55, wherein the antisense compound comprises a nucleobase sequenceselected from SEQ ID NO:1-11.
 60. The method of claim 55, wherein theconnexin target is selected from the group consisting of connexins 45,43, 26, 37, 30 and 31.1.
 61. The method of claim 55, wherein theconnexin target is connexin
 45. 62. The method of claim 55, wherein theconnexin target is connexin
 43. 63. The method of claim 55, wherein theanti-connexin polynucleotide is targeted to at least about 8 nucleobasesof a nucleic acid molecule encoding a connexin.
 64. The method of claim63, wherein the antisense polynucleotide is an antisense oligonucleotideof between about 15 and about 35 nucleobases in length.
 65. The methodof claim 55, wherein the anti-connexin polynucleotide is targeted to atleast about 12 nucleobases of a nucleic acid molecule encoding aconnexin.
 66. The method of claim 55, wherein the anti-connexinpolynucleotide comprises a nucleobase sequence selected from SEQ IDNO:1-11.
 67. The method of claim 55, wherein the anti-connexinpolynucleotide is an antisense oligonucleotide comprising naturallyoccurring nucleobases and an unmodified internucleoside linkage.
 68. Themethod of claim 55, wherein the anti-connexin polynucleotide is anantisense oligonucleotide comprising at least one modifiedinternucleoside linkage.
 69. The method of claim 68, wherein themodified internucleoside linkage is a phosphorothioate linkage.
 70. Themethod of claim 55, wherein the anti-connexin polynucleotide is anoligonucleotide comprising at least one modified sugar moiety.
 71. Themethod of claim 55, wherein the anti-connexin polynucleotide is anoligonucleotide comprising at least one modified nucleobase.
 72. Amethod of claim 55, wherein said anti-connexin polynucleotide isadministered in combination with a second compound useful for reducingtissue damage or promoting healing.
 73. A method of claim 72, whereinthe second compound is a growth factor or cytokine.